Aerosol Jet Printing-Enabled Dual-Function Electrochemical and Colorimetric Biosensor for SARS-CoV-2 Detection.

An aerosol jet printing-enabled dual-function biosensor for the sensitive detection of pathogens using SARS-CoV-2 RNA as an example has been developed. A CRISPR-Cas13:guide-RNA complex is activated in the presence of a target RNA, leading to the collateral trans-cleavage of ssRNA probes that contain a horseradish peroxidase (HRP) tag. This, in turn, catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by HRP, resulting in a color change and electrochemical signal change. The colorimetric and electrochemical sensing protocol does not require complicated target amplification and probe immobilization and exhibits a detection sensitivity in the femtomolar range. Additionally, our biosensor demonstrates a wide dynamic range of 5 orders of magnitude. This low-cost aerosol inkjet printing technique allows for an amplification-free and integrated dual-function biosensor platform, which operates at physiological temperature and is designed for simple, rapid, and accurate point-of-care (POC) diagnostics in either low-resource settings or hospitals.

High-Efficiency Gene Transfection of Cells through Carbon Nanotube Arrays.

Introducing nucleic acids into mammalian cells is a crucial step to elucidate biochemical pathways, and to modify gene expression and cellular development in immortalized cells, primary cells, and stem cells. Current transfection technologies are time consuming and limited by the size of genetic cargo, the inefficient introduction of test molecules into large populations of target cells, and the cytotoxicity of the techniques. A novel method of introducing genes and biomolecules into tens of thousands of mammalian cells has been developed through an array of aligned hollow carbon nanotubes, manufactured by template-based nanofabrication processes, to achieve rapid high-efficiency transfer with low cytotoxicity. The utilization of carbon nanotube arrays for gene transfection overcomes molecular weight limits of current technologies and can be adapted to deliver drugs or proteins in addition to nucleic acids.

Aerosol Jet Printing Enabled Dual-Function Electrochemical and Colorimetric Biosensor for SARS-CoV-2 Detection.

An aerosol jet printing enabled dual-function biosensor for the sensitive detection of pathogens using SARS-CoV-2 RNA as an example has been developed. A CRISPR-Cas13: guide-RNA complex is activated in the presence of a target RNA, leading to the collateral trans-cleavage of ssRNA probes that contain a horseradish peroxidase (HRP) tag. This, in turn, catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by HRP, resulting in a color change and electrochemical signal change. The colorimetric and electrochemical sensing protocol does not require complicated target amplification and probe immobilization and exhibits a detection sensitivity in the femtomolar range. Additionally, our biosensor demonstrates a wide dynamic range of 5 orders of magnitude. This low-cost aerosol inkjet printing technique allows for an amplification-free and integrated dual-function biosensor platform, which operates at physiological temperature and is designed for simple, rapid, and accurate point-of-care (POC) diagnostics in either low-resource settings or hospitals.

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.

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.

Fabrication of nanoscale nozzle for electrohydrodynamic (EHD) inkjet head and high precision patterning by drop-on-demand operation.

Electrohydrodynamic (EHD) spraying has been utilized in applications varying from micro-colloid thrusters to technology for film deposition and inkjet printing. Recently, EHD inkjet heads were developed to facilitate the fabrication of printed electronics such as digital displays, printed circuit boards (PCBs), and solar cells. Here, we report the fabrication and application of nanoscale nozzles for EHD inkjet printing. The nozzles were fabricated by depositing an electrically conductive layer on either the inside or outside of quartz micropipettes with sub-micron diameter tips. With the drop-on-demand control needed for inkjet heads, our nanoscale nozzles dispensed silver droplets in fine patterns on glass substrates.

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.

Single bead-based electrochemical biosensor.

A simple, robust, single bead-based electrochemical biosensor was fabricated and characterized. The sensor's 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 biosensor's 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.

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.

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.

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.

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.