Biosensors and Bioelectronics 26 (2010) 1565–1570 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Integration of microfluidic and cantilever technology for biosensing application in liquid environment  Carlo Ricciardi a,∗ , Giancarlo Canavese b , Riccardo Castagna a , Ivan Ferrante a , Alessandro Ricci a , Simone Luigi Marasso a , Lucia Napione c , Federico Bussolino c a LATEMAR – Politecnico di Torino, Dipartimento di Scienza dei Materiali ed Ingegneria Chimica, Corso Duca degli Abruzzi 24, I-10129 Torino, Italy b IIT – Italian Institute of Technology @ POLITO Center for Human Space Robotics, Corso Trieste 21, 10129 Torino, Italy c Department of Oncological Sciences, Institute for Cancer Research and Treatment, University of Torino, 10060 Candiolo (TO), Italy article info Article history: Received 14 April 2010 Received in revised form 9 July 2010 Accepted 29 July 2010 Available online 5 August 2010 Keywords: Lab-on-Chip Microcantilever Real-time Protein detection Angiogenesis abstract Microcantilever based oscillators have shown the possibility of highly sensitive label-free detection by allowing the transduction of a target mass into a resonant frequency shift. Most of such measurements were performed in air or vacuum environment, since immersion in liquid dramatically deteriorates the mechanical response of the sensor. Besides, the integration of microcantilever detection in a microfluidic platform appears a highly performing technological solution to exploit real time monitoring of biomolec- ular interactions, while limiting sample handling and promoting portability and automation of routine diagnostic tests (Point-Of-Care devices). In the present paper, we report on the realization and opti- mization of a microcantilever-based Lab-on-Chip, showing that microplates rather than microbeams exhibit largest mass sensitivity in liquid, while pirex rather than polymers represents the best choice for microfluidic channels. Maximum Q factor achieved was 140 (for fifth resonance mode of Pirex prototype), as our knowledge the highest value reported in literature for cantilever biosensors resonating in liquid environment without electronic feedback. Then, we proved the successfully detection of Angiopoietin-1 (a putative marker in tumor progression), showing that the related frequency shifts coming from non- specific interactions (negative controls) are roughly one order of magnitude lower than typical variations due to specific protein binding. Furthermore, we monitored the formation of antibody–antigen com- plex on MC surface in real-time. The proposed tool could be extremely useful for the comprehension of complex biological systems such as angiogenic machinery and cancer progression. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Resonance operation method, which aims to quantify the adsorbed target mass thanks to the oscillator frequency shift, seems to be the most successful application of microcantilever (MC) based biosensor. Despite of static operation method (where surface stress generated in binding of the target molecules to the receptors on one MC side cause the beam to deflect), this technique is less affected by the thermal drift of beam deflection and stabilization problems (Shen et al., 2001; Lochon et al., 2006). The progress of microcantilever technology and the need for increasing device sensitivity have favored the reduction of sen- sor dimensions up to the nanoscale. It has been shown that nanocantilevers are able to detect few biomolecules or single viruses (Ilic et al., 2004; Gupta et al., 2004), thus displaying very high mass sensitivity. On the other hand, biosensors at the  Finalist for the World Congress on Biosensors 2010 selected Keynote Paper. ∗ Corresponding author. Tel.: +39 011 0907383. E-mail address: carlo.ricciardi@polito.it (C. Ricciardi). nanoscale have recently shown evident performance limits in terms of analyte density, response time (Nair and Alam, 2006) and statistical variability (Gupta et al., 2006) due to their intrinsic diffusion-limited regime. Furthermore, such highly sensitive can be achieved in air or vacuum environment, since immersion in liquid would dramatically deteriorate the response of the sensor. Indeed, minimum detectable mass can be defined as m min ∝ (m/Q) (Waggoner and Craighead, 2007), where m and Q are the oscilla- tor mass and quality factor, respectively. The latter is defined as Q = f r (f -3 dB ) -1 , where f r is the resonance frequency and f -3 dB is the width of the resonance curve at -3 dB from the maxi- mum. Operation in liquid in fact would be desirable in order to retain biomolecule physiological structure and function, since it has been shown that proteins and cell membrane can change their con- formation passing from liquid environment to vacuum condition (Sharma and Kalonia, 2004). Liquid environment is then funda- mental in those applications where the observation of binding and unbinding kinetics are needed as well as for in situ and real- time measurement. An on-line measurement in liquid can also reduce false positive and false negative responses, which are an 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.07.114