Kinetic Trajectories of Glucose Uptake in Single Cancer Cells Reveal a Drug-Induced Cell-State Change Within Hours of Drug Treatment.

The development of drug resistance is a nearly universal phenomenon in patients with glioblastoma multiforme (GBM) brain tumors. Upon treatment, GBM cancer cells may initially undergo a drug-induced cell-state change to a drug-tolerant, slow-cycling state. The kinetics of that process are not well understood, in part due to the heterogeneity of GBM tumors and tumor models, which can confound the interpretation of kinetic data.

Toward Rapid and Automated Immunoassays: Using a Localized Electrochemical pH Modulation Platform to Perform a Single-Step Immunoassay.

An electrochemical platform for generating and controlling a localized pH microenvironment on demand is proposed by employing a closed-loop control algorithm based on an iridium oxide pH sensor input. We use a combination of solution-borne quinones and galvanostatic excitation on a prepatterned indium tin oxide (ITO) working electrode to modulate pH within a very well confined, small volume of solution close to the electrode surface. We demonstrate that the rate of pH change can be controlled at up to 2 pH s with an excellent repeatability (±0.

Electron-Transport Characteristics through Aluminum Oxide (100) and (012) in a Metal-Insulator-Metal Junction System: Density Functional Theory-Nonequilibrium Green Function Approach.

AlO is commonly used in modern electronic devices because of its good mechanical properties and excellent electrical insulating property. Although fundamental understanding of the electron transport in AlO is essential for its use in electronic device applications, a thorough investigation for the electron-transport mechanism has not been conducted on the structures of AlO, especially in nanometer-scale electronic device settings. In this work, electron transport via AlO for two crystallographic facets, (100) and (012), in a metal-insulator-metal junction configuration is investigated using a density functional theory-based nonequilibrium Green function method.

Intercellular signaling through secreted proteins induces free-energy gradient-directed cell movement.

Controlling cell migration is important in tissue engineering and medicine. Cell motility depends on factors such as nutrient concentration gradients and soluble factor signaling. In particular, cell-cell signaling can depend on cell-cell separation distance and can influence cellular arrangements in bulk cultures.

Single-Cell Phosphoproteomics Resolves Adaptive Signaling Dynamics and Informs Targeted Combination Therapy in Glioblastoma.

Intratumoral heterogeneity of signaling networks may contribute to targeted cancer therapy resistance, including in the highly lethal brain cancer glioblastoma (GBM). We performed single-cell phosphoproteomics on a patient-derived in vivo GBM model of mTOR kinase inhibitor resistance and coupled it to an analytical approach for detecting changes in signaling coordination. Alterations in the protein signaling coordination were resolved as early as 2.

Quantitative assessments of glycolysis from single cells.

The most common positron emission tomography (PET) radio-labeled probe for molecular diagnostics in patient care and research is the glucose analog, 2-deoxy-2-[F-18]fluoro-D-glucose (F-FDG). We report on an integrated microfluidics-chip/beta particle imaging system for F-FDG radioassays of glycolysis with single cell resolution. We investigated the kinetic responses of single glioblastoma cancer cells to targeted inhibitors of receptor tyrosine kinase signaling.

Critical Points in Tumorigenesis: A Carcinogen-Initiated Phase Transition Analyzed via Single-Cell Proteomics.

A kinetic, single-cell proteomic study of chemically induced carcinogenesis is interpreted by treating the single-cell data as fluctuations of an open system transitioning between different steady states. In analogy to a first-order transition, phase coexistence and the loss of degrees of freedom are observed. The transition is detected well before the appearance of the traditional biomarker of the carcinogenic transformation.

Chemical methods for the simultaneous quantitation of metabolites and proteins from single cells.

We describe chemical approaches for integrated metabolic and proteomic assays from single cells. Quantitative assays for intracellular metabolites, including glucose uptake and three other species, are designed as surface-competitive binding assays with fluorescence readouts. This enables integration into a microarray format with functional protein immunoassays, all of which are incorporated into the microchambers of a single-cell barcode chip (SCBC).

Microfluidics-based single-cell functional proteomics for fundamental and applied biomedical applications.

We review an emerging microfluidics-based toolkit for single-cell functional proteomics. Functional proteins include, but are not limited to, the secreted signaling proteins that can reflect the biological behaviors of immune cells or the intracellular phosphoproteins associated with growth factor-stimulated signaling networks. Advantages of the microfluidics platforms are multiple.

Microchip platforms for multiplex single-cell functional proteomics with applications to immunology and cancer research.

Single-cell functional proteomics assays can connect genomic information to biological function through quantitative and multiplex protein measurements. Tools for single-cell proteomics have developed rapidly over the past 5 years and are providing approaches for directly elucidating phosphoprotein signaling networks in cancer cells or for capturing high-resolution snapshots of immune system function in patients with various disease conditions. We discuss advances in single-cell proteomics platforms, with an emphasis on microchip methods.

Hypoxia induces a phase transition within a kinase signaling network in cancer cells.

Hypoxia is a near-universal feature of cancer, promoting glycolysis, cellular proliferation, and angiogenesis. The molecular mechanisms of hypoxic signaling have been intensively studied, but the impact of changes in oxygen partial pressure (pO2) on the state of signaling networks is less clear. In a glioblastoma multiforme (GBM) cancer cell model, we examined the response of signaling networks to targeted pathway inhibition between 21% and 1% pO2.

Quantitating cell-cell interaction functions with applications to glioblastoma multiforme cancer cells.

We report on a method for quantitating the distance dependence of cell-cell interactions. We employ a microchip design that permits a multiplex, quantitative protein assay from statistical numbers of cell pairs, as a function of cell separation, with a 0.15 nL volume microchamber.

A microfluidic-based bubble generation platform enables analysis of physical property change in phospholipid surfactant layers by interfacial ozone reaction.

The air-liquid interface filled with pulmonary surfactant is a unique feature of our lung alveoli. The mechanical properties of this interface play an important role in breathing and its malfunction induced by an environmental hazard, such as ozone, relates to various lung diseases. In order to understand the interfacial physics of the pulmonary surfactant system, we employed a microfluidic bubble generation platform with a model pulmonary surfactant composed of two major phospholipids: DPPC (1,2-dipalmitoyl-sn-phosphatidylcholine) and POPG (1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol).

Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells.

We describe a microchip designed to quantify the levels of a dozen cytoplasmic and membrane proteins from single cells. We use the platform to assess protein-protein interactions associated with the EGF-receptor-mediated PI3K signaling pathway. Single-cell sensitivity is achieved by isolating a defined number of cells (n = 0-5) in 2 nL volume chambers, each of which is patterned with two copies of a miniature antibody array.

A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins.

Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications.

Protein signaling networks from single cell fluctuations and information theory profiling.

Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria.

Time resolved studies of interfacial reactions of ozone with pulmonary phospholipid surfactants using field induced droplet ionization mass spectrometry.

Field induced droplet ionization mass spectrometry (FIDI-MS) comprises a soft ionization method to sample ions from the surface of microliter droplets. A pulsed electric field stretches neutral droplets until they develop dual Taylor cones, emitting streams of positively and negatively charged submicrometer droplets in opposite directions, with the desired polarity being directed into a mass spectrometer for analysis. This methodology is employed to study the heterogeneous ozonolysis of 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol (POPG) at the air-liquid interface in negative ion mode using FIDI mass spectrometry.

Design and optimization of coin-shaped microreactor chips for PET radiopharmaceutical synthesis.

Unlabelled: An integrated elastomeric microfluidic device, with a footprint the size of a postage stamp, has been designed and optimized for multistep radiosynthesis of PET tracers.

Methods: The unique architecture of the device is centered around a 5-microL coin-shaped reactor, which yields reaction efficiency and speed from a combination of high reagent concentration, pressurized reactions, and rapid heat and mass transfer. Its novel features facilitate mixing, solvent exchange, and product collection.

Interfacial reactions of ozone with surfactant protein B in a model lung surfactant system.

Oxidative stresses from irritants such as hydrogen peroxide and ozone (O(3)) can cause dysfunction of the pulmonary surfactant (PS) layer in the human lung, resulting in chronic diseases of the respiratory tract. For identification of structural changes of pulmonary surfactant protein B (SP-B) due to the heterogeneous reaction with O(3), field-induced droplet ionization (FIDI) mass spectrometry has been utilized. FIDI is a soft ionization method in which ions are extracted from the surface of microliter-volume droplets.

A multi-channel electroporation microchip for gene transfection in mammalian cells.

We developed a multi-channel electroporation microchip made of polydimethylsiloxane (PDMS) and glass for gene transfer in mammalian cells. This chip produces multiple electric field gradients in a single microchip by varying the lengths of the microchannels from 2 to 4 cm. Electric fields of 0.

A 160-kilobit molecular electronic memory patterned at 10(11) bits per square centimetre.

The primary metric for gauging progress in the various semiconductor integrated circuit technologies is the spacing, or pitch, between the most closely spaced wires within a dynamic random access memory (DRAM) circuit. Modern DRAM circuits have 140 nm pitch wires and a memory cell size of 0.0408 mum(2).

Quantitative real-time measurements of DNA hybridization with alkylated nonoxidized silicon nanowires in electrolyte solution.

The quantitative, real-time detection of single-stranded oligonucleotides with silicon nanowires (SiNWs) in physiologically relevant electrolyte solution is demonstrated. Debye screening of the hybridization event is circumvented by utilizing electrostatically adsorbed primary DNA on an amine-terminated NW surface. Two surface functionalization chemistries are compared: an amine-terminated siloxane monolayer on the native SiO2 surface of the SiNW, and an amine-terminated alkyl monolayer grown directly on a hydrogen-terminated SiNW surface.

Multistep synthesis of a radiolabeled imaging probe using integrated microfluidics.

Microreactor technology has shown potential for optimizing synthetic efficiency, particularly in preparing sensitive compounds. We achieved the synthesis of an [(18)F]fluoride-radiolabeled molecular imaging probe, 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG), in an integrated microfluidic device. Five sequential processes-[18F]fluoride concentration, water evaporation, radiofluorination, solvent exchange, and hydrolytic deprotection-proceeded with high radio-chemical yield and purity and with shorter synthesis time relative to conventional automated synthesis.

Electrotransfection of mammalian cells using microchannel-type electroporation chip.

Transfection of DNA molecules into mammalian cells with electric pulsations, which is so-called electroporation, is a powerful and widely used method that can be directly applied to gene therapy. However, very little is known about the basic mechanisms of DNA transfer and cell response to the electric pulse. We developed a microelectroporation chip with poly(dimethylsiloxane) (PDMS) to investigate the mechanism of electroporation as a first step of DNA transfer and to introduce the benefits of miniaturization into the genetic manipulation.