Perfusion double-channel micropipette probes for oxygen flux mapping with single-cell resolution.

Measuring cellular respiration with single-cell spatial resolution is a significant challenge, even with modern tools and techniques. Here, a double-channel micropipette is proposed and investigated as a probe to achieve this goal by sampling fluid near the point of interest. A finite element model (FEM) of this perfusion probe is validated by comparing simulation results with experimental results of hydrodynamically confined fluorescent molecule diffusion. The FEM is then used to investigate the dependence of the oxygen concentration variation and the measurement signal on system parameters, including the pipette's shape, perfusion velocity, position of the oxygen sensors within the pipette, and proximity of the pipette to the substrate. The work demonstrates that the use of perfusion double-barrel micropipette probes enables the detection of oxygen consumption signals with micrometer spatial resolution, while amplifying the signal, as compared to sensors without the perfusion system. In certain flow velocity ranges (depending on pipette geometry and configuration), the perfusion flow increases oxygen concentration gradients formed due to cellular oxygen consumption. An optimal perfusion velocity for respiratory measurements on single cells can be determined for different system parameters (e.g., proximity of the pipette to the substrate). The optimum perfusion velocities calculated in this paper range from 1.9 to 12.5 µm/s. Finally, the FEM model is used to show that the spatial resolution of the probe may be varied by adjusting the pipette tip diameter, which may allow oxygen consumption mapping of cells within tissue, as well as individual cells at subcellular resolution.

Separation and liquid chromatography using a single carbon nanotube.

Use of a single template-grown carbon nanotube as a separation column to separate attoliter volumes of binary mixtures of fluorescent dyes has been demonstrated. The cylindrical nanotube walls are used as stationary phase and the surface area is increased by growing smaller multi-walled carbon nanotubes within the larger nanotube column. Liquid-liquid extraction is performed to separate selectively soluble solutes in a solvent, and chromatographic separation is demonstrated using thin, long nanotubes coated inside with iron oxide nanoparticles. The setup is also used to determine the diffusion coefficient of a solute at the sub-micrometer scale. This study opens avenues for analytical chemistry in attoliter volumes of fluids for various applications and cellular analysis at the single cell level.

Physiological validation of cell health upon probing with carbon nanotube endoscope and its benefit for single-cell interrogation.

New-generation nanoscale devices for single-cell study are intensively being developed. As has been shown, nanodevices are minimally invasive because of their order-of-magnitude smaller size in comparison to conventional glass pipettes. However, in most studies the evaluation of the nanodevice impact on cell health has not extended to their effects on cell metabolic integrity. In this work we evaluated the degree to which the insertion of a carbon-based nanotube endoscope into a cell induces mechanical and biochemical stress, and affects cellular key metabolic systems. The effects of insertion of the nanotube endoscope on cell morphological and physiological modulations were monitored and compared to those of glass micropipettes. We report that nanotube endoscope insertion does not significantly modulate the plasma membrane and actin network. The cell metabolic mechanisms such as energy production and inositol 1,4,5-trisphosphate-dependent calcium signaling remain preserved for prolonged endoscope presence within a cell. FROM THE CLINICAL EDITOR: In this basic science study, the effects of insertion of carbon nanotube endoscope on cell morphological and physiological modulations were monitored and compared to those of glass micropipettes. Nanotube endoscope insertion is truly minimally invasive: it does not significantly modulate the plasma membrane and actin network; the energy production and inositol 1,4,5-trisphosphate-dependent calcium signaling also remain preserved during prolonged endoscope presence within a cell.

Multifunctional carbon-nanotube cellular endoscopes.

Glass micropipettes, atomic force microscope tips and nanoneedles can be used to interrogate cells, but these devices either have conical geometries that can damage cells during penetration or are incapable of continuous fluid handling. Here, we report a carbon-nanotube-based endoscope for interrogating cells, transporting fluids and performing optical and electrochemical diagnostics at the single organelle level. The endoscope, which is made by placing a multiwalled carbon nanotube (length, 50-60 µm) at the tip of a glass pipette, can probe the intracellular environment with a spatial resolution of ∼100 nm and can also access organelles without disrupting the cell. When the nanotube is filled with magnetic nanoparticles, the endoscope can be remotely manoeuvered to transport nanoparticles and attolitre volumes of fluids to and from precise locations. Because they are mounted on conventional glass micropipettes, the endoscopes readily fit standard instruments, creating a broad range of opportunities for minimally invasive intracellular probing, drug delivery and single-cell surgery.

Small diameter carbon nanopipettes.

Nanoscale multifunctional carbon probes facilitate cellular studies due to their small size, which makes it possible to interrogate organelles within living cells in a minimally invasive fashion. However, connecting nanotubes to macroscopic devices and constructing an integrated system for the purpose of fluid and electrical signal transfer is challenging, as is often the case with nanoscale components. We describe a non-catalytic chemical vapor deposition based method for batch fabrication of integrated multifunctional carbon nanopipettes (CNPs) with tip diameters much smaller (10-30 nm) than previously reported (200 nm and above) and approaching those observed for multiwalled carbon nanotubes. This eliminates the need for complicated attachment/assembly of nanotubes into nanofluidic devices. Variable tip geometries and structures were obtained by controlled deposition of carbon inside and outside quartz pipettes. We have shown that the capillary length and gas flow rate have a marked effect on the carbon deposition. This gives us a flexible protocol, useful for growing carbon layers of different thicknesses at selective locations on a glass pipette to yield a large variety of cellular probes in bulk quantities. The CNPs possess an open channel for fluid transfer with the carbon deposited inside at 875 degrees C behaving like an amorphous semiconductor. Vacuum annealing of the CNP tips at temperatures up to 2000 degrees C yields graphitic carbon structures with an increase in conductivity of two orders of magnitude. Penetration of the integrated carbon nanoprobes into cells was shown to produce minimal Ca(2+) signals, fast recovery of basal Ca(2+) levels and no adverse activation of the cellular metabolism during interrogation times as long as 0.5-1 h.