John M. Nagarah

Piezoelectric nanoribbons for monitoring cellular deformations

Methods for probing mechanical responses of mammalian cells to electrical excitations can improve our understanding of cellular physiology and function. The electrical response of neuronal cells to applied voltages has been studied in detail, but less is known about their mechanical response to electrical excitations. Studies using atomic force microscopes (AFMs) have shown that mammalian cells exhibit voltage-induced mechanical deflections at nanometre scales, but AFM measurements can be invasive and difficult to multiplex. Here we show that mechanical deformations of neuronal cells in response to electrical excitations can be measured using piezoelectric PbZr(x)Ti(1-x)O(3) (PZT) nanoribbons, and we find that cells deflect by 1 nm when 120 mV is applied to the cell membrane. The measured cellular forces agree with a theoretical model in which depolarization caused by an applied voltage induces a change in membrane tension, which results in the cell altering its radius so that the pressure remains constant across the membrane. We also transfer arrays of PZT nanoribbons onto a silicone elastomer and measure mechanical deformations on a cow lung that mimics respiration. The PZT nanoribbons offer a minimally invasive and scalable platform for electromechanical biosensing.

Wafer-Scale Nanopatterning and Translation into High-Performance Piezoelectric Nanowires

The development of a facile method for fabricating one-dimensional, precisely positioned nanostructures over large areas offers exciting opportunities in fundamental research and innovative applications. Large-scale nanofabrication methods have been restricted in accessibility due to their complexity and cost. Likewise, bottom-up synthesis of nanowires has been limited in methods to assemble these structures at precisely defined locations. Nanomaterials such as PbZr(x)Ti(1-x)O(3) (PZT) nanowires (NWs)--which may be useful for nonvolatile memory storage (FeRAM), nanoactuation, and nanoscale power generation--are difficult to synthesize without suffering from polycrystallinity or poor stoichiometric control. Here, we report a novel fabrication method which requires only low-resolution photolithography and electrochemical etching to generate ultrasmooth NWs over wafer scales. These nanostructures are subsequently used as patterning templates to generate PZT nanowires with the highest reported piezoelectric performance (d(eff) ∼ 145 pm/V). The combined large-scale nanopatterning with hierarchical assembly of functional nanomaterials could yield breakthroughs in areas ranging from nanodevice arrays to nanodevice powering.

Fast Nonlinear Ion Transport via Field-Induced Hydrodynamic Slip in Sub-20-nm Hydrophilic Nanofluidic Transistors

Electrolyte transport through an array of 20 nm wide, 20 microm long SiO(2) nanofluidic transistors is described. At sufficiently low ionic strength, the Debye screening length exceeds the channel width, and ion transport is limited by the negatively charged channel surfaces. At source-drain biases >5 V, the current exhibits a sharp, nonlinear increase, with a 20-50-fold conductance enhancement. This behavior is attributed to a breakdown of the zero-slip condition. Implications for energy conversion devices are discussed.

Ultradeep fused silica glass etching with an HF-resistant photosensitive resist for optical imaging applications

Microfluidic and optical sensing platforms are commonly fabricated in glass and fused silica (quartz) because of their optical transparency and chemical inertness. Hydrofluoric acid (HF) solutions are the etching media of choice for deep etching into silicon dioxide substrates, but processing schemes become complicated and expensive for etching times greater than 1 h due to the aggressiveness of HF migration through most masking materials. We present here etching into fused silica more than 600 µm deep while keeping the substrate free of pits and maintaining a polished etched surface suitable for biological imaging. We utilize an HF-resistant photosensitive resist (HFPR) which is not attacked in 49% HF solution. Etching characteristics are compared for substrates masked with the HFPR alone and the HFPR patterned on top of Cr/Au and polysilicon masks. We used this etching process to fabricate suspended fused silica membranes, 8–16 µm thick, and show that imaging through the membranes does not negatively affect image quality of fluorescence microscopy of biological tissue. Finally, we realize small through-pore arrays in the suspended membranes. Such devices will have applications in planar electrophysiology platforms, especially where optical imaging is required.

Batch Fabrication of High‐Performance Planar Patch‐Clamp Devices in Quartz

The success of the patch-clamp technique has driven an effort to create wafer-based patch-clamp platforms. We develop a lithographic/electrochemical processing scheme that generates ultrasmooth, high aspect ratio pores in quartz. These devices achieve gigaohm seals in nearly 80% of trials, with the majority exhibiting seal resistances from 20-80 GΩ, competing with pipette-based patch-clamp measurements.

Optically transparent multi-suction electrode arrays.

Multielectrode arrays (MEAs) allow for acquisition of multisite electrophysiological activity with submillisecond temporal resolution from neural preparations. The signal to noise ratio from such arrays has recently been improved by substrate perforations that allow negative pressure to be applied to the tissue; however, such arrays are not optically transparent, limiting their potential to be combined with optical-based technologies. We present here multi-suction electrode arrays (MSEAs) in quartz that yield a substantial increase in the detected number of units and in signal to noise ratio from mouse cortico-hippocampal slices and mouse retina explants. This enables the visualization of stronger cross correlations between the firing rates of the various sources. Additionally, the MSEAs transparency allows us to record voltage sensitive dye activity from a leech ganglion with single neuron resolution using widefield microscopy simultaneously with the electrode array recordings. The combination of enhanced electrical signals and compatibility with optical-based technologies should make the MSEA a valuable tool for investigating neuronal circuits.

Neurons on a Chip – Toward High Throughput Network and Pharmacology Investigations

The biophysical properties of single neurons and synaptic connections between populations of neurons enable the brain to form memories, make decisions, and perceive the surrounding environment. The ability of neurons to transmit signals is reliant on ion channel proteins embedded in the plasma membrane (Hodgkin and Huxley, 1952; Koch, 2005). Ion channel defects and neural network dysfunction are the basis of several neurological disorders (Ackerman and Clapham, 1997; Palop et al., 2006; Seeley et al., 2009). The development of new technologies, such as those mentioned within this issue, is fueled by the desire not only to understand the fundamental processes underlying neuronal signals, but also, to gain a better understanding of the causes, and thus potential treatments for neurological diseases. Traditionally, ion channel protein activity in single cells has been monitored using the glass pipette patch-clamp technique (Hamill et al., 1981). Our detailed understanding of ion channel kinetics and pharmacological properties made possible by this technique is one of the reasons why so many drug targets include ion channels (Wang and Li, 2003). However, the patch-clamp technique has been limited by the number of cells from which it can simultaneously record. Recent advances in robotics have enabled measurements in brain slices from 12 simultaneous pipette patch-clamp electrodes, illustrating novel electrophysiological principles that govern network behavior, though these experiments are still demanding on the user (Anastassiou et al., 2011). In contrast, multi-electrode arrays (MEAs) and silicon-based transistor arrays allow for extracellular recordings from hundreds and thousands of electrodes simultaneously (Pine, 1980; Hutzler et al., 2006). Despite the limited amount of biophysical information obtained from these experiments, the high throughput of MEAs has enabled their successful use in discovering novel network mechanisms (Hosoya et al., 2005) and in screening potential pharmaceutical compounds (Hill et al., 2010; Redon et al., 2010). As each technique presents its own strengths and weaknesses, combining the throughput of the MEA platform with the resolution and control of the patch-clamp would provide a unique hybrid that would be highly welcomed in many areas of neuroscience. Recently, researchers have succeeded in replacing the glass pipette with a micro-machined orifice in a planar substrate. Through their ability to achieve an electrical resistance greater than 1 gigaohm between the cell membrane and pore, planar patch-clamp electrodes have enabled high quality electrophysiology recordings (Fertig et al., 2002; Nagarah et al., 2010). Automated patch-clamp (APC) systems have been used primarily for pharmaceutical screening against ion channels of interest expressed in cell lines. Only recently have APCs been used to investigate primary cells or physiologically more relevant cells derived from pluripotent stem cells (Milligan et al., 2009; Stoelzle et al., 2011). In this issue, Py et al. (2011) describe their success in integrating cultured neurons onto planar patch-clamp electrodes. A significant accomplishment of theirs is obtaining gigaohm seals between poly-l-lysine (PLL) coated apertures and cultured neurons. This is surprising considering the contamination susceptibility of patch-clamp electrodes, suggesting the sealing mechanism of suspended cells is different than that of cultured neurons. Their platform has allowed for intracellular recordings of neuronal activity from cultured neurons and synapse formation on the chip, demonstrated by dual planar and pipette measurements along with pharmacological characterization. Simultaneous recordings from synaptically connected neurons with dual planar electrodes have proven difficult so far, but nonetheless, they are able to record from multiple apertures simultaneously using an improved device fabrication scheme and microfluidic channels. The authors are also making advancements in chemical patterning methods to precisely localize neurons onto the planar orifices which will eliminate the need for manual placement. While the bulk of recordings have been from snail neurons, the authors have recently integrated mammalian neurons onto their device, increasing the applicability of this platform to drug screening. High throughput pharmaceutical screening on patch-clamped neuronal cultures will enable the observation of a drugs effect on synaptic transmission as well as ion channel behavior. Additionally, the planar nature of the devices described here opens up the possibility of integrating with it other technologies. For example, a novel microfluidic culture system has allowed for investigations of the cell biology of synaptic functions with high spatial and temporal resolution (Taylor et al., 2010). Combining a similar system with planar patch-clamp electrodes would add electrophysiological control to cell biology experiments, and provide even more detail to synaptic dynamics investigations. Further development is still needed before such experiments are made possible, but Py et. al.’s efforts will undoubtedly contribute to the growing list of tools that neuroscientists use to provide insight in neural computation, the nature of neurological diseases, and the treatment of diseases.