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. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells

Single cell phosphoproteomics identifies adaptive network dynamics of mTOR inhibitor resistance and defines effective combination therapy in glioblastoma

Resistance to single-agent targeted cancer therapy is almost universal. Resistance can occur when drug-resistant tumor cell subpopulations expand to drive recurrence in a process akin to Darwinian-type evolution under the selection pressure of the drug. An alternative resistance mechanism is the one in which cancer cells targeted by the inhibitor adapt to that drug, so as to maintain the signal flux through those networks that are required for tumor maintenance and growth. The main goal of this study is to identify the mechanisms of resistance in a targeted therapy by analyzing single cells and to provide a strategy to design a more effective therapy that suppresses resistance

Formation of ZnO Micro-Flowers Prepared via Solution Process and their Antibacterial Activity

This paper presents the fabrication and characterization of zinc oxide micro-flowers and their antibacterial activity. The micro-flowers of zinc oxide composed of hexagonal nanorods have been prepared via solution process using precursor zinc acetate di-hydrate and sodium hydroxide in 3 h of refluxing time at ~90°C. The antibacterial activities of grown micro-flowers were investigated against four pathogenic bacteria namely S. aureus, E. coli, S. typhimurium and K. pneumoniae by taking five different concentrations (5–45 μg/ml) of ZnO micro-flowers (ZnO-MFs). Our investigation reveals that at lowest concentration of ZnO-MFs solution inhibiting the growth of microbial strain which was found to be 5 μg/ml for all the tested pathogens. Additionally, on the basis of morphological and chemical observations, a chemical reaction mechanism of ZnO-MFs composed of hexagonal nanorods was also proposed

Synthesis, characterization and effect of pH variation on zinc oxide nanostructures

Here we present a systematic study on the morphological deviation of ZnO nanostructure (from sheets to micro-flowers) by varying pH of the solution via precipitation method. In this regard, zinc nitrate hexa-hydrate, NaOH and hydroxylamine hydrochloride (NH 2 OHÁHCl) were used. The solution of all three compounds was refluxed at a very low temperature (60 C) for short time (20 min). The solution pH was calibrated from 6 to 12 by the controlled addition of NaOH and HCl. We have observed from FESEM (field emission scanning electron microscopy) that the morphology of ZnO microballs composed with thin sheets markedly varies from sheet (at pH ¼ 6) to micro-flower composed with sheets of zinc oxide (pH ¼ 10{12). Further the morphology and crystallinity were also studied by the TEM (transmission electron microscopy) and HR-TEM (High resolution transmission electron microscopy) and it's clearly consistent with the FESEM observations. The FTIR spectroscopic measurement also confirms the compositional analysis of ZnO and it comes in the range of 475 to 424 cm À1 which is a standard peak of ZnO. In addition to this, the amount of H þ and OH À ions are found a key to control the structure of studied material and discussed in the growth mechanism

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. Here, we seek a physical-based approach, which identifies a potential governed by cell–cell signaling that induces a directed cell–cell motion. A single-cell barcode chip (SCBC) was used to experimentally interrogate secreted proteins in hundreds of isolated glioblastoma brain cancer cell pairs and to monitor their relative motions over time. We used these trajectories to identify a range of cell–cell separation distances where the signaling was most stable. We then used a thermodynamics-motivated analysis of secreted protein levels to characterize free-energy changes for different cell–cell distances. We show that glioblastoma cell–cell movement can be described as Brownian motion biased by cell–cell potential. To demonstrate that the free-energy potential as determined by the signaling is the driver of motion, we inhibited two proteins most involved in maintaining the free-energy gradient. Following inhibition, cell pairs showed an essentially random Brownian motion, similar to the case for untreated, isolated single cells

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. First, 20 or more functional proteins may be assayed simultaneously from statistical numbers of single cells. Second, cell behaviors (e.g., motility) may be correlated with protein assays. Third, extensions to quantized cell populations can permit measurements of cell–cell interactions. Fourth, rare cells can be functionally identified and then separated for further analysis or culturing. Finally, certain assay types can provide a conduit between biology and the physicochemical laws. We discuss the history and challenges of the field then review design concepts and uses of the microchip platforms that have been reported, with an eye toward biomedical applications. We then look to the future of the field

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). With fluorescence imaging, we observed the ozone-induced chemical modification of the unsaturated lipid component of the lipid mixture, POPG. This chemical change due to the oxidative stress was further utilized to study the physical characteristics of the interface through the bubble formation process. The physical property change was evaluated through the oscillatory behaviour of the monolayer, as well as the bubble size and formation time. The results presented demonstrate the potential of this platform to study interfacial physics of lung surfactant system under various environmental challenges, both qualitatively and quantitatively

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. We report structurally specific oxidative changes of SP-B_(1−25) (a shortened version of human SP-B) at the air−liquid interface. We also present studies of the interfacial oxidation of SP-B_(1−25) in a nonionizable 1-palmitoyl-2-oleoyl-sn-glycerol (POG) surfactant layer as a model PS system, where competitive oxidation of the two components is observed. Our results indicate that the heterogeneous reaction of SP-B_(1−25) at the interface is quite different from that in the solution phase. In comparison with the nearly complete homogeneous oxidation of SP-B_(1−25), only a subset of the amino acids known to react with ozone are oxidized by direct ozonolysis in the hydrophobic interfacial environment, both with and without the lipid surfactant layer. Combining these experimental observations with the results of molecular dynamics simulations provides an improved understanding of the interfacial structure and chemistry of a model lung surfactant system subjected to oxidative stress