Michael Gabi

FluidFM: Combining Atomic Force Microscopy and Nanofluidics in a Universal Liquid Delivery System for Single Cell Applications and Beyond

We describe the fluidFM, an atomic force microscope (AFM) based on hollow cantilevers for local liquid dispensing and stimulation of single living cells under physiological conditions. A nanofluidic channel in the cantilever allows soluble molecules to be dispensed through a submicrometer aperture in the AFM tip. The sensitive AFM force feedback allows controlled approach of the tip to a sample for extremely local modification of surfaces in liquid environments. It also allows reliable discrimination between gentle contact with a cell membrane or its perforation. Using these two procedures, dyes have been introduced into individual living cells and even selected subcellular structures of these cells. The universality and versatility of the fluidFM will stimulate original experiments at the submicrometer scale not only in biology but also in physics, chemistry, and material science.

Force-controlled spatial manipulation of viable mammalian cells and micro-organisms by means of FluidFM technology

The FluidFM technology uses microchanneled atomic force microscope cantilevers that are fixed to a drilled atomic force microscope cantilevers probeholder. A continuous fluidic circuit is thereby achieved extending from an external liquid reservoir, through the probeholder and the hollow cantilever to the tip aperture. In this way, both overpressure and an underpressure can be applied to the liquid reservoir and hence to the built-in fluidic circuit. We describe in this letter how standard atomic force microscopy in combination with regulated pressure differences inside the microchanneled cantilevers can be used to displace living organisms with micrometric precision in a nondestructive way. The protocol is applicable to both eukaryotic and prokaryotic cells (e.g., mammalian cells, yeasts, and bacteria) in physiological buffer. By means of this procedure, cells can also be transferred from one glass slide to another one or onto an agar medium.

Electrochemically switchable platform for the micro-patterning and release of heterotypic cell sheets

This article describes a dynamic platform in which the biointerfacial properties of micro-patterned domains can be switched electrochemically through the spatio-temporally controlled dissolution and adsorption of polyelectrolyte coatings. Insulating SU-8 micro-patterns created on a transparent indium tin oxide electrode by photolithography allowed for the local control over the electrochemical dissolution of polyelectrolyte mono- and multilayers, with polyelectrolytes shielded from the electrochemical treatment by the underlying photoresist stencil. The platform allowed for the creation of micro-patterned cell co-cultures through the electrochemical removal of a non-fouling polyelectrolyte coating and the localized adsorption of a cell adhesive one after attachment of the first cell population. In addition, the use of weak adhesive polyelectrolyte coatings on the photoresist domains allowed for the detachment of a contiguous heterotypic cell sheet upon electrochemical trigger. Cells grown on the ITO domains peeled off upon electrochemical dissolution of the sacrificial polyelectrolyte substrate, whereas adjacent cell areas on the insulated weakly adhesive substrate easily detached through the contractile force generated by neighboring cells. This electrochemical strategy for the micro-patterning and detachment of heterotypic cell sheets combines simplicity, precision and versatility, and presents great prospects for the creation of cellular constructs which mimic the cellular complexity of native tissues.

Electrically controlling cell adhesion, growth and migration.

We have developed a neurochip to control the adhesion and outgrowth of individual neurons by electrochemical removal of protein repellent molecules from transparent electrodes. The neurochip architecture is based on three parallel indium-tin-oxide (ITO) electrodes on a SiO(2) substrate and a photoresist structure forming a landing spot for the neuron soma and two lateral outgrowth pathways for the neurites. The whole surface was turned protein and cell repellent with poly(ethylene glycol) grafted-poly(L-lysine) (PLL-g-PEG) before enabling neuron soma adhesion by selective PLL-g-PEG removal. After the neuron has settled down a potential was applied to the pathway electrodes to permit the neurite outgrowth along pathways formed by the SU8 structure. We also show the possibility to control cell migration by small pulsed currents. Myoblasts were therefore seeded on a chemical pattern of cell adhesive PLL and cell resistant PLL-g-PEG. The PLL-g-PEG was then removed electrochemically from the electrodes to permit migration onto the cell free electrodes. Electrodes without applied current were confluently overgrown within 24 h but a small pulsed current was able to inhibit cell growth on the bare ITO electrode for more than 72 h. With both techniques, cell adhesion, growth and migration can be controlled dynamically after the cells started to grow on the substrate. This opens new possibilities: we believe the key to control the development of topologically controlled neuron networks or more complex co-cultures is the combination of passive surface modifications and active control over the surface properties at any time of the experiment.

Electrical microcurrent to prevent conditioning film and bacterial adhesion to urological stents

Long-term catheters remain a significant clinical problem in urology due to the high rate of bacterial colonization, infection, and encrustation. Minutes after insertion of a catheter, depositions of host urinary components onto the catheter surface form a conditioning film actively supporting the bacterial adhesion process. We investigated the possibility of reducing or avoiding the buildup of these naturally forming conditioning films and of preventing bacterial adhesion by applying different current densities to platinum electrodes as a possible catheter coating material. In this model we employed a defined environment using artificial urine and Proteus mirabilis. The film formation and desorption was analyzed by highly mass sensitive quartz crystal microbalance and surface sensitive atomic force microscopy. Further, we performed bacterial staining to assess adherence, growth, and survival on the electrodes with different current densities. By applying alternating microcurrent densities on platinum electrodes, we could produce a self regenerative surface which actively removed the conditioning film and significantly reduced bacterial adherence, growth, and survival. The results of this study could easily be adapted to a catheter design for clinical use.

Enhanced optical waveguide light mode spectroscopy via detection of fluorophore absorbance

A novel technique based on surface sensitive absorbance detection using an optical waveguide light mode spectroscopy (OWLS) instrument is presented. The proof of concept for this extension of a standard technique is demonstrated by painting an increasing number of ink lines on a waveguide, perpendicular to the light path, while monitoring the outcoupled light intensity. Furthermore, by the adsorption of poly(L-lysine)-graft-poly(ethylene glycol) as a model system with contents of 5%, 10%, 25%, and 50% labeled polymer, the in situ performance is demonstrated, and the absorbance signal is calibrated such that it can be converted into adsorbed mass. The simultaneous detection of labeled and label-free species allows for the study of complex experimental setups whereby monitoring of adsorption, desorption, and even exchange processes becomes possible. The sensitivity of the absorbance detection exceeds standard OWLS by one to two orders of magnitude.

Effects of small pulsed nanocurrents on cell viability in vitro and in vivo: Implications for biomedical electrodes

Using a custom-built, implantable pulse generator, we studied the effects of small pulsed currents on the viability on rat aortic-derived cells (RAOC) in vitro. The pulsed currents (0.37A/m(2)) underwent apoptosis within 24h as shown by the positive staining for cleaved caspase-3 and classically apoptotic morphology. Based on these findings, we examined the effects of nanocurrents in vivo. The pulse generator was implanted subcutaneously in the rat model. The electrode|tissue interface histology revealed no difference between the active platinum surface and the neighboring control surface, however we found a large difference between electrodes that were functional during the entire experiment and non-active electrodes. These non-active electrodes showed an increase in impedance at higher frequencies 21 days post-implantation, whereas working electrodes retained their impedance value for the entire experiment. These results indicate that applied currents can reduce the impedance of implanted electrodes.