FluidFM: Combining AFM and nanofluidics for single cell applications

The Atomic Force Microscope (AFM) is a key tool for nanotechnology. This instrument has become the most widely used tool for imaging, measuring and manipulating matter at the nanoscale and in turn has inspired a variety of other scanning probe techniques. Originally the AFM was used to image the topography of surfaces, but by modifying the tip it is possible to measure other quantities (for example, electric and magnetic properties, chemical potentials, friction and so on), and also to perform various types of spectroscopy and analysis. Increasingly, the AFM is also becoming a tool for nanofabrication.

Relatively new is the use of AFM in cell biology. We wrote about this recently in a Spotlight that described a novel method to probe the mechanical properties of living and dead bacteria via AFM indentation experimentations ("Dead or alive – nanotechnology technique tells the difference ").
Researchers in Switzerland have now demonstrated novel cell biology applications using hollow force-controlled AFM cantilevers – a new device they have called FluidFM.

"The core of the invention is to have fixed already existing microchanneled cantilevers to an opportunely drilled AFM probeholder" Tomaso Zambelli tells Nanowerk. "In this way, the FluidFM is not restricted to air but can work in liquid environments. Since it combines a nanofluidics circuit, every soluble agent can be added to the solution to be dispensed. Moreover, the force feedback allows to approach very soft objects like cells without damaging them."

As cell biology is moving towards single cell technologies and applications, single cell injection or extraction techniques are in high demand. Apart from this, however, the FluidFM could also be used for nanofabrication applications such as depositing a conductive polymer wire between to microelectrodes, or to etch ultrafine structures out of solid materials using acids as the spray agent. The team has reported their findings in a recent paper in Nano Letters ("FluidFM: Combining Atomic Force Microscopy and Nanofluidics in a Universal Liquid Delivery System for Single Cell Applications and Beyond").

Staining living neuroblastoma cells by gentle contact on the cell membrane. (a) Diagram showing the staining procedure by gentle contact (not to scale). The hollow tip is maintained in contact with the cell membrane thanks to the force feedback (set point of less than 1 nN). The active agents dissolved in the solution of the microchannel spontaneously diffuse across the membrane into the cytoplasm. (b) Superposition of a differential interference contrast image and of the corresponding fluorescent one of a cell after staining with CellTracker green. The microchannelled cantilever filled with CellTracker green is positioned over the cell in the red circle using the optical microscope. The tip is then brought into gentle contact with the cell membrane by the AFM force feedback system and left there for 15 min, before taking the fluorescent and phase contrast image

Zambelli originally realized that the technology of the atomic force microscope that is normally used only to image cells could be transformed into a microinjection system. The result of the development by Zambelli and his colleagues in the Laboratory of Biosensors and Bioelectronics at the Institute of Biomedical Technology at ETH Zurich and in the Swiss Center for Electronics and Microtechnology (CSEM) in Neuchâtel was the "fluid force microscope", currently the smallest automated nanosyringe currently in existence.

"Our FluidFM even operates under water or in other liquids – a precondition for being able to use the instrument to study cells" says Zambelli.

The force detection system of the FluidFM is so sensitive that the interactions between tip and sample can be reduced to the piconewton range, thereby allowing to bring the hollow cantilever into gentle but close contact with cells without puncturing or damaging the cell membrane.

On the other hand, if membrane perforation for intracellular injection is desired, this is simply achieved by selecting a higher force set point taking advantage of the extremely sharp tip (radius of curvature on the order of tens of nanometers).

To enable solutions to be injected into the cell through the needle, scientists at CSEM installed a microchannel in the cantilever. Substances such as medicinal active ingredients, DNA, and RNA can be injected into a cell through the tip. At the same time, samples can also be taken from a cell through the needle for subsequent analysis.

According to Zambelli, while this approach is similar to microinjection using glass pipettes, there are a number of essential differences.

"Microinjection uses optical microscopy to control the position of the glass pipette tip both in the xy plane and in the z direction (via image focusing)" he explains. "As consequence of the limited resolution of optical microscopy, subcellular domains cannot be addressed and tip contact with the cell membrane cannot be discriminated from tip penetration of the membrane. Cells are often lethally damaged and skilled personnel are required for microinjection."

"The limited resolution of this method and the absence of mechanical information contrast strongly with the high resolution imaging and the direct control of applied forces that are possible with AFM. Precise force feedback reduces potential damage to the cell; the cantilever geometry minimizes both the normal contact forces on the cell and the lateral vibrations of the tip that can tear the cell membrane during microinjection; the spatial resolution is determined by the submicrometer aperture so that injection into subcellular domains becomes easily achievable."

Experiments conducted by the Swiss team demonstrate the potential of the FluidFM in the field of single-cell biology through precise stimulation of selected cell domains with whatever soluble agents at a well-defined time.

"We confidently expect that the inclusion of an electrode in the microfluidics circuit will allow a similar approach toward patch-clamping with force controlled gigaseal formation," says Zambelli. "We will also explore other strategies at the single-cell level, such as the controlled perforation of the cell membrane for local extraction of cytoplasm

"Zambelli and his colleagues are convinced that their technology has great commercial potential. Rejecting offers from well-known manufacturers of atomic force microscopes for the sale of the patent for the FluidFM, they have founded Cytosurge LLC, a company dedicated to commercially develop the instrument.

Today, Zambelli's laboratory contains two prototypes of the instrument, which are being tested in collaboration with biologists.

Nanofluidics meets nanoplasmonic sensing

Nanofluidics meets nanoplasmonic sensing

In physics, a plasmon is the quasiparticle resulting from the quantization of plasma oscillations just as photons and phonons are quantizations of light and sound waves, respectively. As the name indicates, surface plasmons are those plasmons that are confined to surfaces. Controlling these surface plasmons has become increasingly attractive for optical signal processing, surface enhanced spectroscopy and sensor nanotechnology. For instance, the role of surface plasmon resonance (SPR) on resonant transmission through nanohole arrays has motivated their application as surface-based biosensors.

"As compared to common SPR sensing, nanohole arrays present many advantages, including a smaller foot-print, lower limits of detection, denser integration, multiplexing, and collinear optical detection," David Sinton, an associate professor in the Department of Mechanical Engineering at the University of Victoria in Canada, explains to Nanowerk. "Arrays of nanoholes in a metal film exhibit unique optical properties in part due to the presence of surface plasmons – light waves confined near the metallic surface. Due to the confinement of the light near the surface, nanohole arrays have been employed to sense the binding of biological molecules, with application to biosensing."

As an example, Sinton says that cancer screening may be accomplished using suitably coated nanohole array based sensors. One of the problems so far has been to transport an analyte of interest to a surface based sensor, and thus achieve a low limit of detection.

New work by a team of scientists from Sinton's, Brolo's and Gordon's group at the University of Victoria, and the British Columbia Cancer Agency, has combined nanofluidics and nanoplasmonics for SPR sensing using flow-through nanohole arrays. This new format enables rapid transport of reactants to the active sensing surface and the array serves as a sieve. That is, the flow-through array efficiently collects and detects biomarkers from a very small volume of fluid.

Schematic of the flow-through nanohole arrays and fluorescence image showing a dye buffer solution streaming from the array.

Sinton points out that their findings indicate a 6-fold improvement in response time as compared to the established method.

The team has reported their findings in a recent paper in Analytical Chemistry ("Nanoholes As Nanochannels: Flow-through Plasmonic Sensing").

"Research to date has focused on arrays of dead-ended holes, or pits" says Sinton. "In terms of transport, previous nanohole array sensors didn't take advantage of their nanostructure. Our findings indicate that flow-through nanohole array format offers significant advantages with respect to the transport and sieving of biomarkers."

The researchers fabricated their circular nanohole arrays by focused-ion beam milling through a 100 nm thick gold film thermally evaporated on free-standing 100 nm silicon nitride with a 5 nm thick chromium adhesion layer. By varying the milling parameters they were able to determine the conditions for fabricating arrays that allowed through-hole transport.

This work has been motivated by the need for an effective cancer screening method where quantitative detection of multiple biomarkers is required in order to provide a diagnosis. Particularly motivating are such diseases that are treatable if detected early, yet generally asymptomatic until late stages. An example is ovarian cancer as studied by collaborators at the BC Cancer Trev & Joyce Deeley Research Centre.

Sinton explains how the new device's combination of nanofluidics and nanoplasmonics works: "As fluidic elements, the nanohole arrays serve to parallelize the resistance and thus fluid handling and control are compatible between the parallel nanochannels and established microfluidic protocols. As optical elements, the nanohole arrays serve to detect the adsorption of a monolayer as well as the step-by-step multilayer assembly of biomolecules as required for biosensing applications."

Sinton suggests that future work will focus on two areas. "Firstly, flow-through nanohole array based sensors have the potential for dense integration in a lab-on-chip format, but the implementation of that is not straightforward and is a current focus. Secondly, the transition from microscale to nanoscale channels that is involved with this new flow-through format offers some interesting physics. There are several potential opportunities to exploit these fundamental phenomena to achieve fluid pumping, analyte concentration and control of particles in the vicinity of the sensing surface with implications for sensing."