Sensors and Actuators A 139 (2007) 162–171 Glass-based microfluidic device fabricated by parylene wafer-to-wafer bonding for impedance spectroscopy Daniel P. Poenar a,∗ , Ciprian Iliescu b,1 , Mihaela Carp a , Ah Ju Pang b,1 , Kwong Joo Leck b,1 a Microelectronics Centre, School of Electrical & Electronical Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore b Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore Received 28 July 2006; received in revised form 8 September 2006; accepted 6 October 2006 Available online 1 November 2006 Abstract The paper reports the realization of a glass-based microfluidic device for characterization of cells in suspensions using impedance spectroscopy. The device consists of two glass wafers: a bottom wafer comprising a microfluidic channel with two electrodes added for impedance measurement, and a top glass wafer in which inlets and outlets are realized. The main focus of this work is the original fabrication process of this device, which combines three key techniques: firstly, successfully applying a through-wafer wet etching method in order to pattern the inlets and outlets in the top glass wafer; secondly, patterning the electrodes not only on flat surfaces but also in the microfluidic channel etched in the bottom wafer; and thirdly, employing indirect wafer to wafer bonding using an intermediate polymer layer. No external pumping is required as capillarity enables direct suction of a sample droplet in the channel. The device can be reusable if a thorough cleaning procedure is carried out. Devices with three different electrode geometries were successfully tested in electrochemical impedance spectroscopy (EIS) measurements using DI water and HepG2 cells. Although clear differences between DI water and live HepG2 cells have been observed in all cases, different results were obtained for various electrode geometries, highlighting the critical importance of the device design in performing EIS measurements and especially when making comparisons with other reported results. © 2006 Elsevier B.V. All rights reserved. Keywords: Microfluidics; Thru-wafer HF etching of glass; Spray coating photolithography; Parylene bonding; Electrochemical impedance spectroscopy (EIS); HepG2 cells 1. Introduction A starting point in our work was the need to find out a cell characterization method more convenient than classical ones, such as dielectrophoresis or cell immobilization. Dielectrophoresis (DEP) is a very well-known method with wide applications in the manipulation, separation and charac- terization of bio-components. It is based on creating an electric field gradient, e.g. by using electrodes of different shapes that can induce a change in the electric field magnitude across the par- ticle. These electrodes can be thin films [1], bulk electrodes [2,3] or even a combination of bulk and thin electrodes [4]. A related method – traveling wave DEP (TWD) – consists of changing ∗ Corresponding author. Tel.: +65 67904237; fax: +65 67920415. E-mail address: EPDPuiu@ntu.edu.sg (D.P. Poenar). 1 Tel.: +65 68247137; fax: +65 64789082. the phase of the applied electric field [5] while in the so-called “isolating DEP” (iDEP) the electric field gradient is generated by a non-homogenous dielectric medium placed between the electrodes [6]. Nevertheless, for all these DEP versions, due to the high concentration of electric field lines in certain locations, care must be taken in the design and usage of such devices for bio-applications in order to prevent cell lysis or other possi- ble structural damage (e.g. electroporation in the case of cells). Indeed, it is possible and was practically demonstrated that DEP- based cell separation and cell lysis can be very conveniently integrated together in a single unit [7]. Moreover, the application of DEP for cell separation relies on the a priori knowledge of the exact dielectric properties of all the bio-components present in the specific sample to be ana- lyzed. This is necessary in order to apply the correct frequency (or range of frequencies) and voltage which would allow the efficient separation or identification of the desired component. If this information is not available, the researcher has to carry 0924-4247/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.sna.2006.10.009