Michael G. Schrlau

Essential requirement for two-pore channel 1 in NAADP-mediated calcium signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a widespread and potent calcium-mobilizing messenger that is highly unusual in activating calcium channels located on acidic stores. However, the molecular identity of the target protein is unclear. In this study, we show that the previously uncharacterized human two-pore channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of TPC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release by NAADP. Thus, TPC1 is critical for NAADP action and is likely the long sought after target channel for NAADP.

Cell Electrophysiology with Carbon Nanopipettes

The ability to monitor living cell behavior in real time and with high spatial resolution is vital for advancing our knowledge of cellular machinery and evaluating cellular response to various drugs. Here, we report the development and utilization of carbon-based nanoelectrodes for cell electrophysiology. We employ carbon nanopipettes (CNPs), novel carbon-based nanoprobes which integrate carbon nanopipes into the tips of pulled glass capillaries, to measure electrical signals in the mouse hippocampal cell line HT-22. Using a standard electrophysiology amplifier in current-clamp mode, we measured the resting membrane potential of cells and their transient membrane response to extracellular pharmacological agents. In addition to their superior injection capabilities reported previously, CNPs are capable of multifunctionality, enabling, for example, concurrent intracellular injection and electrical measurements without damaging cells.

Carbon nanopipettes for cell probes and intracellular injection

We developed integrated, carbon-based pipettes with nanoscale dimensions (CNP) that can probe cells with minimal intrusion, inject fluids into the cells, and concurrently carry out electrical measurements. Our manufacturing technique does not require cumbersome nanoassembly and is amenable to mass production. Using CNPs, we demonstrate the injection of reagents into cells with minimal intrusion and without inhibiting cell growth.

Polymeric microbead arrays for microfluidic applications

Microbeads offer a convenient and efficient means of immobilizing biomolecules and capturing target molecules of interest in microfluidic immunoassay devices. In this study, hot embossing is used to form wells enabling the direct incorporation of a microbead array in a plastic substrate. We demonstrate two techniques to populate the well array with beads. In the first case, encoded beads with various functionalizations are distributed randomly among the wells and their position is registered by reading their encoding. Alternatively, beads are controllably placed at predetermined positions and decoding is not required. The random placement technique is demonstrated with two functionalized bead types that are distributed among the wells and then decoded to register their locations. The alternative, deliberate placement technique is demonstrated by controllably placing magnetic beads at selected locations in the array using a magnetic probe. As a proof of concept to illustrate the biosensing capability of the randomly assembled array, an on-chip, bead-based immunoassay is employed to detect the inflammatory protein Interleukin-8. The principle of the assay, however, can be extended to detect multiple targets simultaneously. Our method eliminates the need to interface silicon components with plastic devices to form microarrays containing individually addressable beads. This has the potential to reduce the cost and complexity of lab-on-chip devices for medical diagnosis, food and water quality inspection, and environmental monitoring.

Carbon nanotube-tipped endoscope for in situ intracellular surface-enhanced Raman spectroscopy.

Gold nanoparticle-decorated carbon nanotubes (CNTs) are used to study intracellular environments in situ using surface-enhanced Raman spectroscopy (SERS). CNTs are decorated with gold nanoparticles and assembled onto the tips of pulled glass capillaries to form a SERS-enabled endoscope. The sub-micrometer size and high mechanical strength of the endoscope make it possible to penetrate the cell membrane for intracellular probing and remain positioned inside during lengthy SERS measurements without causing damage to the cell. Using the SERS-enabled endoscope, DNA and other biomolecules are detected in situ within the nucleus of a single human cervical carcinoma cell in a minimally invasive manner. The SERS-enabled endoscopes exhibit high selectivity and sensitivity for detecting trace amounts of analytes (≈1 pM) in biofluid environments, highlighting their capabilities as label-free, biological sensors for real-time in situ cellular diagnostics, biological detection, and pharmaceutical research.

Carbon nanopipettes characterize calcium release pathways in breast cancer cells.

Carbon-based nanoprobes are attractive for minimally invasive cell interrogation but their application in cell physiology has thus far been limited. We have developed carbon nanopipettes (CNPs) with nanoscopic tips and used them to inject calcium-mobilizing messengers into cells without compromising cell viability. We identify pathways sensitive to cyclic adenosine diphosphate ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP) in breast carcinoma cells. Our findings demonstrate the superior utility of CNPs for intracellular delivery of impermeant molecules and, more generally, for cell physiology studies. The CNPs do not appear to cause any lasting damage to cells. Their advantages over commonly used glass pipettes include smaller size, breakage and clogging resistance, and potential for multifunctionality such as in concurrent injection and electrical measurements.

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

UNLABELLED 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.

Single bead-based electrochemical biosensor.

A simple, robust, single bead-based electrochemical biosensor was fabricated and characterized. The sensors working electrode consists of an electrochemically etched platinum wire, with a nominal diameter of 25 microm, hermetically heat-fusion sealed in a pulled glass capillary (micropipette). The sealing process does not require any epoxy or glue. A commercially available, densely functionalized agarose bead was mounted on the tip of the etched platinum wire. The use of a pre-functionalized bead eliminates the tedious and complicated surface functionalization process that is often the bottleneck in the development of electrochemical biosensors. We report on the use of a biotin agarose bead-based, micropipette, electrochemical (Bio-BMP) biosensor to monitor H(2)O(2) concentration and the use of a streptavidin bead-based, micropipette, electrochemical (SA-BMP) biosensor to detect DNA amplicons. The Bio-BMP biosensors response increased linearly as the H(2)O(2) concentration increased in the range from 1 x 10(-6) to 1.2 x10(-4)M with a detection limit of 5 x 10(-7)M. The SA-BMP was able to detect the amplicons of 1pg DNA template of B. Cereus bacteria, thus providing better detection sensitivity than conventional gel-based electropherograms.

Carbon Nanotube-Based Sensors: Overview

Since the 1990s, carbon nanotubes (CNTs) and related nanostructures have inspired the engineering, scientific, and medical communities to utilize their novel characteristics and superior properties in a variety of applications. Carbon nanostructures can help strengthen materials and composites, further miniaturize electronics, and enable more efficient green energy technology. Many of the exciting possibilities of carbon nanostructures exist in sensing applications, where their unique properties can be applied to improve the detection of biological threats, efficiently and compactly monitor environmental conditions and screen the health of patients, and detect the early onset of disease. Carbon nanostructures come in a variety of sizes, shapes, and configurations, which make them well suited for a broad range of sensing applications. This chapter focuses on the development of several CNT-based sensing platforms and recount the current utilization and application trends in biological sensing.

Carbon Nanopipettes for Cell Surgery

Carbon nanopipettes (CNPs), nanoprobes that integrate carbon nanotubes (CNTs) into larger easily maneuverable devices, provide a viable means of performing cell surgery with carbon-based nanostructures. Taking advantage of the nanoscopic tubular geometry and unique material properties of CNTs, CNPs facilitate minimally invasive cell probing, low-volume intracellular fluid injection, sensitive electrical measurements of cell signals, and other unique analytical abilities not possible with traditional glass-based cell probing technology. In this technology review, we highlight the cell probing applications where CNPs were used as nanoneedles for intracellular injection and nanoelectrodes for cell electrophysiology. Besides summarizing the proven capabilities of CNPs, the intent of this review is to encourage further development of CNT-based devices and related nanotechnology for novel cell probing and bioanalytical applications.