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Biological functionalization of massively parallel arrays of nanocantilevers using microcontact printing

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Abstract

In this paper, we present a back-end method for biofunctionalizing a large-scale array of nanocantilevers. Our method relies on the use of a modified microcontact printing process where molecules are delivered onto the fragile structures from the grooves of the stamp while its base sits on the chip, thus providing mechanical stability. We have used this method to print antibodies onto fabricated chips containing up to 105 nanostructures/cm2and the presence of antibodies was validated by fluorescent microscopy. Furthermore, measurement of the nanocantilever resonant frequency shifts provoked by a mean added mass of ∼140 fg/cantilever demonstrated that the cantilevers retained their mechanical integrity. Hence, the method presented here aims at providing an answer to the biofunctionalization of freestanding nanostructures for their use as biosensors.

Introduction

One of the most promising applications of nanoelectromechanical systems (NEMS) is foreseen in the field of ultrasensitive mechanical biosensing [1], [2], [3], [4]. For this to become reality, a major challenge is the functionalization of closely packed nanostructures [5] in such a way that biological receptors are precisely located solely onto the active biosensing areas, thus preventing the waste of biological matter and enabling the subsequent biological blocking of the passive parts of the chip. So far, the issue of the freestanding nanostructures functionalization has been seldom addressed because of the absence of generic tools or techniques allowing large-scale molecular delivery at the nanoscale. One way to circumvent this difficulty is to perform the functionalization step before completing the fabrication of the NEMS. This strategy can typically be used in a top-down NEMS fabrication process by protecting the biological layer during the subsequent NEMS fabrication steps, that consists in placing the functionalized nanostructures at specific locations on a substrate and releasing them [6], [7]. The main limitation of this strategy is the tradeoff between the choice of the post-functionalization processing steps and the resilience of the chosen biological receptors to such technological constraints, which for most of them are biologically unfriendly.

In this paper, we report on the use of a corrugated polymer stamps technique [8] for the post-process biological functionalization of large arrays of freestanding nanocantilevers while preserving their mechanical integrity. Thermally oxidized silicon nanocantilevers are fabricated and then functionalized with fluorescently labeled immunoglobulin (IgG) using a specially designed microcontact printing (μCP) stamp. The presence of IgG onto the desired locations is validated both by fluorescent microscopy and measurement of nanocantilevers quality factor and resonant frequency shifts provoked by the corresponding added mass.

Section snippets

Materials and methods

Arrays of nanocantilevers were fabricated using silicon-on-insulator (SOI) substrates and a UV stepper photo repeater. The use of SOI wafers (340 nm P-type Si/1 μm SiO2/525 μm Si from Soitec, France) ensured the production of cantilevers with a controllable thickness (since fabricated in the top silicon layer) and the release of the cantilevers in aqueous solution without structure collapsing and sticking issues (by using the 1 μm-thick buried SiO2 as a sacrificial layer). A UV stepper photo

Results and discussion

After printing the antibodies, the array of nanocantilevers were imaged using an Olympus IX70 inverted fluorescence microscope, a 20× objective (N.A. 0.40, Olympus), a dedicated filter (U-M41008, Olympus) and a Clara CCD ANDOR camera (DR-328G-C01-SIL, 10 s acquisition time). Fluorescence images of arrays of functionalized NEMS are shown in Fig. 3. The high fluorescence signal to noise ratio demonstrate that the IgG were indeed deposited at the desired location, i.e. onto the nanocantilevers.

Conclusion

In conclusion we have presented a method for biofunctionalizing a large-scale array of nanocantilevers using a modified μCP process. Our biofunctionalization technique enables to pattern probe molecules used for biorecognition events only onto the nanostructures, thus allowing the reduction of biological waste. More importantly, by subsequently coating the non-reactive areas of the chip with an anti-fouling film to lower the probability of adsorption of target molecules anywhere else than on

Acknowledgements

The French National Agency for Research (program ANR/PNANO 2008, project NEMSPIEZOANR-08-NANO-015’) is gratefully acknowledged for financial support.

Samuel Guillon is a Ph.D. student in the NanoElectroMechanical Structures field at the Laboratory for Analysis and Architecture of Systems and the University of Toulouse, France. He obtained his Master degree in Materials and Nanotechnology from the French National Institute of Applied Sciences, Rennes, in 2008.

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Samuel Guillon is a Ph.D. student in the NanoElectroMechanical Structures field at the Laboratory for Analysis and Architecture of Systems and the University of Toulouse, France. He obtained his Master degree in Materials and Nanotechnology from the French National Institute of Applied Sciences, Rennes, in 2008.

Sven Salomon received his Ph.D. degree from the University of Toulouse III, France in 2011. He obtained his M.S. from the INPG, France in 2008.

Florent Seichepine graduated from the ENSIACET engineer school; he received his Ph.D. from the Université de Toulouse in 2011. He works on the large-scale integration of carbon nanotubes for applications in microelectronics and tissues engineering and on the large-scale integration of nano-resonators.

Denis Dezest is a Ph.D. student at the CNRS Laboratory for Analysis and Architecture of Systems in Toulouse, France. He obtained his M.S. in Engineering Physics from the National Institute of Applied Sciences of Toulouse in 2010.

Fabrice Mathieu was born in Orléans, France, in 1972. He received the Engineer degree in communication systems and electronics from the CNAM (Conservatoire National des Arts et Métiers), Toulouse, France, in 2003. He joined the Laboratoire d’Analyse et d’Architecture des Systèmes (LAAS), Centre National de la Recherche Scientifique (CNRS), Toulouse, in 2001, where he is currently in charge of the development and the design of very low signal detection systems applied to the micro(nano)electromechanical systems area and its complete electronic treatment and control for automation.

Aude Bouchier is a research engineer at the Laboratory for Analysis and Architecture of Systems in Toulouse (France). She received the Master of Science from the Paris XI University (France), the Master in Optics from the Institut d’Optique Graduate School (France) in 2002, and her Ph.D. degree in laser physics in 2005 from Paris XI University (France).

Laurent Mazenq was born on May 30, 1982. He received his University Institute of Technology's Degree from the University Paul Sabatier de Toulouse (France) in 2002. Then, he joined the Laboratoire d’Analyse et d’Architecture des Systèmes of the French Centre National de la Recherche Scientifique (LAAS-CNRS) as an Engineer Assistant. In 2011, he joined Freescale Semiconductor (France) in production process Engineering. He is working on UV projection lithography.

Christophe Thibault is an assistant professor at the Laboratory for Analysis and Architecture of Systems in Toulouse, France. He obtained his Master degree from University of Toulouse III, in 2004 and his Ph.D. from the University of Toulouse in 2007.

Christophe Vieu is professor at INSA, National Institute of Applied Science, University of Toulouse, and researcher at the Laboratory for Analysis and Architecture of Systems of CNRS organization. He received his Ph.D. in solid-state physics from the University of Toulouse in 1987. His first achievements were dedicated to the fabrication of semiconductor nanostructures and single electron devices. His current research activity is focused on nanobiotechnologies, nanomedicine and nanoeducation.

Thierry Leïchlé is a CNRS researcher at the Laboratory for Analysis and Architecture of Systems in Toulouse (France). He obtained his M.S. from the Georgia Institute of Technology, Atlanta (USA), in 2002 and his Ph.D. from the University of Toulouse III in 2006. From 2007 to 2009 he was a postdoctoral fellow at the Institute of Physics, Academia Sinica, Taipei (Taiwan).

Liviu Nicu was born in 1973 in Bucharest (Romania). After completing his Master of Electrical Engineering at the Paul Sabatier University of Toulouse (France) in 1997, he joined the Integrated Microsystems Group at the LAAS (Laboratory for Analysis and Architecture of Systems) of Toulouse where he obtained his Ph.D. in 2000 into the Micromechanical Structures field. From 2000 to 2003, he was R&D Engineer at Thales Avionics, Valence (France). His activities focused onto the development of micromechanical sensors for the civil and military navigation applications. Since 2003 he joined the NanoBioSytems Group at LAAS as a full time CNRS (National Center of Scientific Research) researcher where he currently works in two main research fields: the development of (1) new resonant bio(chemical)sensors using M(N)EMS technologies and of (2) cantilever-based microsystems for contact deposition of small amounts of biological samples for biochip applications.

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