Modified cantilever arrays improve sensitivity and reproducibility of nanomechanical sensing in living cells

Mechanical signaling involved in molecular interactions lies at the heart of materials science and biological systems, but the mechanisms involved are poorly understood. Here we use nanomechanical sensors and intact human cells to provide unique insights into the signaling pathways of connectivity networks, which deliver the ability to probe cells to produce biologically relevant, quantifiable and reproducible signals. We quantify the mechanical signals from malignant cancer cells, with 10 cells per ml in 1000-fold excess of non-neoplastic human epithelial cells. Moreover, we demonstrate that a direct link between cells and molecules creates a continuous connectivity which acts like a percolating network to propagate mechanical forces over both short and long length-scales. The findings provide mechanistic insights into how cancer cells interact with one another and with their microenvironments, enabling them to invade the surrounding tissues. Further, with this system it is possible to understand how cancer clusters are able to co-ordinate their migration through narrow blood capillaries.

of the total surface area. A control cantilever had 100 µm wide strips running centrally along the entire of its length, and corresponding to 100% of the total surface area. SAMs of mercaptoundecanoic acid (MUA) and mercaptohexadecanoic acid (MHA) were used to create transduction arrays. MUA was used to create 30 µm wide strips for both parallel and transverse arrays as well as for control cantilevers. MHA was used to create 50 µm strips in the parallel arrays as well as for control nanomechanical cantilevers. These SAMs were chosen because of their capacity to be attached to a variety of receptor molecules, allowing the detection of a diversity of ligands. For example, in this study, MUA SAMs were attached to the VSR and anti-IgG antibody to enable detection of vancomycin (Van) and immunoglobulin G (IgG) respectively. Further, these SAMs are alkanethiols with less than 20 carbons and are known to enable stable printing patterns with defined boundaries 4 and by functionalizing mechanical sensors with SAMs of carboxylic terminating groups, the scaffolds can be switched between 'oxidized' and 'reduced' states when the pH of the surrounding environment is changed 5 . To block nonspecific interactions, the unpatterned areas on the cantilever surface were passivated using SAMs of undecanethiol (UDT) in the case of MUA, and hexadecanethiol (HDT) for MHA.

Preparation of silicon substrates
Silicon substrates measuring 1 cm x 1 cm each was cleaned by incubating in a freshly prepared piranha solution, consisting of H 2 SO 4 and H 2 O 2 (1:1) for 20 min. They were then briefly rinsed in ultrapure water followed by a rinse in pure ethanol before drying on a hotplate at 75 °C. The silicon substrates were examined under an optical microscope to confirm their cleanliness before transferring to an electron beam evaporation chamber (BOC Edwards Auto 500, U.K.) where they were coated at a rate of 0.7 nms -1 with a 2 nm layer of titanium, which act as an adhesion layer, followed by a 20 nm layer of gold. Once the required thickness of gold was obtained, the silicon substrates were left in the chamber for 1-2 h to cool under vacuum.

Printing of transduction arrays µCP stamps
Transduction arrays were printed on the gold-coated silicon substrates using SAMs of MUA as the printing ink. The protocol for printing MUA SAMs onto gold-coated silicon substrates is summarized in Supplementary Figure 1 Having established that the dip-pen nanolithography could be successfully used to write the array patterns on gold-coated silicon substrates, we then applied the same procedure to create transduction arrays on the nanomechanical cantilevers.

Location specific mechanical force in cell response
In Supplementary Figure 4

Derivation of expression for binding interactions
We first proposed that solvent effects are dominant factors important in predetermining mechanical forces exerted by cells or molecules. Accordingly, we considered that an analyte can interact with a surface target and so the reactions are quantified by considering the distributions between analyte's concentrations in where n is the stoichiometric coefficient of the reaction and analyte.R is the bound complex. The expression between an analyte and receptor is where K d is the surface thermodynamic equilibrium dissociation constant. The total concentration of the surface receptor [R] T is Using equations (2) and (3)

Analysis mechanical force
The differential stress measurements obtained from cantilever chips are typically associated with multiple parameters including the number of repeated measurements, concentration and the number of cantilevers where each has eight individual cantilever arrays. The differential equilibrium mechanical forces for a wide range of concentrations of analytes (0.0005 -1000 µM) or cells (10 -1000 cells ml -1 ) were determined using 4 chips for each ligand. For each analyte concentration, the arithmetic mean of the differential mechanical force data (F eq ) across 4 chips was calculated using the equation The standard error (SE) of the differential mechanical force for each analyte concentration was calculated using the equation