Sensors and Actuators B 176 (2013) 1026–1031 Contents lists available at SciVerse ScienceDirect Sensors and Actuators B: Chemical journa l h o me pa ge: www.elsevier.com/locate/snb Leveraging on nanomechanical sensors to single out active small ligands for  2 -microglobulin Giulio Oliviero a , Marcella Chiari b , Ersilia De Lorenzi c , Raffaella Colombo c , Marina Cretich b , Francesco Damin b , Stefania Federici a , Laura E. Depero a,∗∗ , Paolo Bergese a,∗ a Chemistry for Technologies Laboratory and INSTM, University of Brescia, Via Branze 38, 25123 Brescia, Italy b Istituto di Chimica del Riconoscimento Molecolare (ICRM), C.N.R., Via Mario Bianco, 9, 20131, Milano, Italy c Department of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy a r t i c l e i n f o Article history: Received 18 July 2012 Received in revised form 4 September 2012 Accepted 11 September 2012 Available online 25 September 2012 Keywords: Small ligands 2-microglobulin Conformational changes Fibrillogenesis Nanomechanics Microcantilevers a b s t r a c t A nanomechanical biosensor based on microcantilevers was implemented to test low molecular weight (small) compounds for their ability to stabilize  2 -microglobulin ( 2 -m) in its native conformation.  2 -m was immobilized on the top face of silicon microcantilevers and it was demonstrated that pH induced unfolding of the immobilized  2 -m drives a specific microcantilever bending. This  2 -m microcantilever assay was then implemented to probe the effect of a pilot set small ligands on  2 -m conformational stability. Among the tested ligands, congo red was the only one able to protect  2 -m from unfolding, that is known to be the primary trigger of its self-polymerization into fibrils and in turn of the onset of amyloidosis. These findings disclose the high potentiality of nanomechanical sensors in the field of protein conformation related diseases, as they bring the unique advantage of directly screening com- pounds for their specific pharmacological activity rather than for generic binding preferences, effectively shortcutting the identification of the active ones. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Beta 2 -microglobulin ( 2 -m) is a protein responsible for the onset of the dialysis related amyloidosis (DRA), a severe and unavoidable complication that occurs in patients undergoing chronic hemodialysis, where insoluble and toxic  2 -m amyloid deposits (fibrils) localize in the skeletal tissues [1]. As for all amyloidoses, the progressive self-polymerization of  2 -m monomer into fibrils follows a complex and still unclear mechanism, where protein conformational changes, among other factors, are a key event. Over the past 10 years, by using spec- troscopic probes including stopped flow fluorescence [2], circular dichroism [3,4], NMR [5,6], dynamic light scattering [7], mass spectrometry [8,9] as well as separation techniques like capillary electrophoresis [10,11], an increasing wealth of information has been accumulated on the understanding of both  2 -m folding and amyloid assembly mechanisms. ∗ Corresponding author. Tel.: +39 030 3715667; fax: +39 030 3702448. ∗∗ Corresponding author. Tel.: +39 030 3715472; fax: +39 030 3702448. E-mail addresses: giulio.oliviero@ing.unibs.it (G. Oliviero), marcella.chiari@icrm.cnr.it (M. Chiari), ersidelo@unipv.it (E. De Lorenzi), raffaella.colombo@unipv.it (R. Colombo), marina.cretich@icrm.cnr.it (M. Cretich), francesco.damin@icrm.cnr.it (F. Damin), stefania.federici@ing.unibs.it (S. Federici), laura.depero@ing.unibs.it (L.E. Depero), paolo.bergese@ing.unibs.it (P. Bergese). It has been reported that small molecules can modify the kinet- ics of amyloid fibrils formation [12,13] as well as the equilibrium between native and partially folded protein species.  2 -m is a chal- lenging target, since it is a small globular protein (99 aminoacids) that lacks a specific binding site. Our studies carried out over the past decade by affinity capillary electrophoresis and SPR [11,14,15] and high resolution mass spectrometry [16,17] have selected a panel of sulfonated molecules whose binding to  2 -m affects its refolding kinetics and its in vitro anti-amyloid activity. It is still unknown whether the measured activity is induced by the preven- tion of conformational changes upon binding, as it was recently established to be the case for doxycycline [18]. In this work we address this open issue by implementing ad- hoc  2 -m nanomechanical biosensors, as they are uniquely suited to probe protein conformational changes. They are based on the fact that surface transformations of biomolecules come along with molecular nanoscale motions that drive peculiar molecule–surface and molecule–molecule interactions. These forces can cumulate and trigger a surface work (pressure) of mJ/m 2 [19] that can be probed by CONtact Angle Molecular Recognition (CONAMORE) [20] or by the nanomechanical effects it drives on MEMS (MicroElec- tro Mechanical Systems) of proper shape [21,22]. The transduction mechanism is therefore directly linked to the energetic and mechanical aspects of the biomolecular transformation rather than to the mass and/or the optical density of the biomolecules that undergo the transformation, in contrast with other current 0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2012.09.032