Parallel AFM imaging and force spectroscopy using two-dimensional probe arrays for applications in cell biology

Atomic force microscopy (AFM) investigations of living cells provide new information in both biology and medicine. However, slow cell dynamics and the need for statistically significant sample sizes mean that data collection can be an extremely lengthy process. We address this problem by parallelizing AFM experiments using a two-dimensional cantilever array, instead of a single cantilever. We have developed an instrument able to operate a two-dimensional cantilever array, to perform topographical and mechanical investigations in both air and liquid. Deflection readout for all cantilevers of the probe array is performed in parallel and online by interferometry. Probe arrays were microfabricated in silicon nitride. Proof-of-concept has been demonstrated by analyzing the topography of hard surfaces and fixed cells in parallel, and by performing parallel force spectroscopy on living cells. These results open new research opportunities in cell biology by measuring the adhesion and elastic properties of a large number of cells. Both properties are essential parameters for research in metastatic cancer development. Copyright (C) 2011 John Wiley & Sons, Ltd.

Stimuli-responsive nanostructured surfaces

Ana Maria Popa

The coupling of stimuli-responsive macromolecules to nanostructured surfaces opens the perspective for the fabrication and integration of "smart" micro- and nano-electro-mechanical systems (MEMS&NEMS) in which the smallest motile unit is the polymeric chain. The goal of this work was to develop thermoresponsive, durable nanoporous surfaces as a first step toward the fabrication of functional nanocontainers and nanovalves. The proposed strategy involves in a first step the design and implementation of a clean-room compatible, reproducible and up-scalable method for defining organized nanoporous arrays in MEMS compatible surfaces, such as silicon, silicon oxide and silicon nitride. The main objective of the second step was the covalent immobilization of responsive macromolecules on the obtained structures, as to provide the later with a "smart" behavior. The nanostructuring step was realized by block-copolymer assisted lithography. This newly developed technique enables the definition of nanoscale features on semiconductor substrates over large surface areas and in a cost effective manner. A novel method for patterning thin metal films by a lift-off approach, using spin coated block-copolymer micelles monolayers as a template, was investigated. The obtained nanoporous mask was used as resist for the structuring of hard substrates in plasma-assisted processes and structures with aspect ratios as high as 1:10 were be obtained. By using different block-copolymer micelles and spin-coating conditions, the proposed approach enables the variation of the nanopores' diameter from 40 to 80 nm and a surface coverage between 11.7% and 25.5 % in an independent manner. This technique was successfully integrated in a standard microfabrication process for the batch production of thin nanoporous silicon nitride membranes. The 100 nm thick membranes span over thousands of square micrometers and present porosities higher than 109 pores/cm2. They are thus competitive in terms of porosity with the commercially available polymeric membranes. In addition they are two orders of magnitude thinner than polymeric membranes, this making them extremely interesting for applications where permeation rate or response time is important. The rapid flow of water soluble small molecular species through the nanopores was demonstrated in a qualitative manner in this work. Another series of investigations targeted the functionalization of the available nanoporous surfaces with responsive molecules. Poly(N-isopropyl acrylamide) (PNIPAAM) was chosen as a model system. Its coupling to silicon surfaces by a "grafting-to" approach from melt, using an epoxy-functionalized silane as anchoring layer was studied. The analysis of the yielded grafting density showed results superior to those obtained by classic grafting-to approaches. The relation between the polymer's weight and its responsiveness was investigated in liquid media by atomic force microscopy and by dynamic force spectroscopy. Polymers with more than 500 monomers in the back bone were shown to exhibit enhanced responsiveness. Additionally, two bioengineered polymers from the class of elastin-like polymers (ELPs) were investigated as possible candidates for the fabrication of stimuli responsive nanovalves. The molecules were synthesized in collaboration with the Bioforge group (Universidad de Valladolid, Spain) and details of the bioproduction process are presented. A method for the covalent photoattachment of macromolecules using a benzophenone-functionalized silane, was tested in this work. First the capacity of the benzophenone silane to graft synthetic thermo-responsive polymers such as PNIPAAM was demonstrated. The temperature-induced morphology change of the photografted PNIPAAM was not affected by this anchoring strategy and was monitored by tapping AFM in liquid media. The investigations were then extended to the photo-immobilization of ELPs on nanoporous silicon surfaces and the XPS measurements revealed the success of this step. However no conformation modification of the proteins could be evidenced by tapping-mode AFM in liquid media under different ionic strengths.

EPFL2009

Integrated long-range thermal bimorph actuators for parallelizable bio-AFM applications

Jürgen Brugger, Rosario Maurizio Gullo, Jonas Gustav Henriksson

AFM-based cell force spectroscopy is an emerging research method that already has enhanced our understanding of the structural changes that take place in a cell as it becomes cancerous. However, the method is limited as it is not time-efficient in its current state of development. This paper presents the fabrication of an integrated long-range thermal bimorph actuator that controls the z-position of an AFM cantilever in liquid. Multiplied in arrays, such individually actuated probes can parallelize cell force spectroscopy measurements, thereby drastically reducing the time per measured cell. The need to accommodate differences in tip-sample distance implies an individual device actuation range of ≥10 µm out of plane. In addition, any cross-talk, i.e. between actuators or between the actuator and the force sensor, must be minimized. To meet these requirements, we designed and fabricated a novel thermal bimorph actuator that was incorporated with force sensing cantilevers. In order to keep temperatures in a bio-friendly range, the design was optimized for high thermo-mechanical sensitivity. FEM simulations confirmed that the surrounding liquid constitutes a large thermal reservoir that absorbs the generated heat. Furthermore, given that a cell substrate material of high thermal conductivity is chosen, in our case silicon, the thermal coupling between the cell and the substrate dominates over that between the cell and the actuator. Suspended silicon nitride structures with platinum electrodes were micro-fabricated through standard techniques. The finalized actuator was able to displace the cantilever out of plane by 17 µm in air.

Institute of Electrical and Electronics Engineers2013

Integrated long-range thermal bimorph actuators for parallelizable bio-AFM applications

Jürgen Brugger, Rosario Maurizio Gullo, Jonas Gustav Henriksson

Ieee2012

Stimuli-responsive nanostructured surfaces

Ana Maria Popa

EPFL2009