Young Shik Shin

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. Multiple doses of [18F]FDG for positron emission tomography imaging studies in mice were prepared. These results, which constitute a proof of principle for automated multistep syntheses at the nanogram to microgram scale, could be generalized to a range of radiolabeled substrates.

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. The SiNWs without the native oxide exhibit improved solution-gated field-effect transistor characteristics and a significantly enhanced sensitivity to single-stranded DNA detection, with an accompanying 2 orders of magnitude improvement in the dynamic range of sensing. A model for the detection of analyte by SiNW sensors is developed and utilized to extract DNA-binding kinetic parameters. Those values are directly compared with values obtained by the standard method of surface plasmon resonance (SPR) and demonstrated to be similar. The nanowires, however, are characterized by higher detection sensitivity. The implication is that SiNWs can be utilized to quantitate the solution-phase concentration of biomolecules at low concentrations. This work also demonstrates the importance of surface chemistry for optimizing biomolecular sensing with silicon nanowires.

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. The cells are lysed on-chip, and the levels of released proteins are assayed using the antibody arrays. We investigate three isogenic cell lines representing the cancer glioblastoma multiforme, at the basal level, under EGF stimulation, and under erlotinib inhibition plus EGF stimulation. The measured protein abundances are consistent with previous work, and single-cell analysis uniquely reveals single-cell heterogeneity, and different types and strengths of protein-protein interactions. This platform helps provide a comprehensive picture of altered signal transduction networks in tumor cells and provides insight into the effect of targeted therapies on protein signaling networks.

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 2.5 days after treatment, anticipating drug resistance long before it was clinically manifest. Combination therapies were identified that resulted in complete and sustained tumor suppression in vivo. This approach may identify actionable alterations in signal coordination that underlie adaptive resistance, which can be suppressed through combination drug therapy, including non-obvious drug combinations.

We have developed a microchip for polymerase chain reaction (PCR) with polydimethylsiloxane (PDMS). PDMS has good characteristics: it is cheap, transparent, easy to fabricate and biocompatible. But in micro PCR, the porosity of PDMS causes several critical problems such as bubble formation, sample evaporation and protein adsorption. To solve those problems, we coated the micro PCR chips with Parylene film, which has low permeability to moisture and long-term stability. We investigated the influence of low thermal conductivity of PDMS and Parylene on the thermal characteristics of the PCR chips with numerical analysis. The thermal responses of micro PCR chips were compared for three materials: silicon, glass and PDMS. From the results, we identified appropriate thermal responses of the PDMS-based micro PCR chips by heating both the top and bottom sides. We could successfully amplify the angiotensin converting enzyme gene with as small a volume as 2 μl on the PDMS-based micro PCR chips without any additives.

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). By using the SCBC, we interrogate the response of human-derived glioblastoma cancer cells to epidermal growth factor receptor inhibition. We report, for the first time, on both the intercellular metabolic heterogeneity as well as the baseline and drug-induced changes in the metabolite-phosphoprotein correlation network.

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. <b>Methods:</b> The unique architecture of the device is centered around a 5-μL 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. New mixing mechanisms assisted by vacuum, pressure, and chemical reactions are exploited. <b>Results:</b> The architecture of the reported reactor is the first that has allowed batch-mode microfluidic devices to produce radiopharmaceuticals of sufficient quality and quantity to be validated by in vivo imaging. <b>Conclusion:</b> The reactor has the potential to produce multiple human doses of ¹⁸F-FDG; the most impact, however, is expected in the synthesis of PET radiopharmaceuticals that can be made only with low yields by currently available equipment.

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. We interrogate interactions between pairs of model brain cancer cells by assaying for six functional proteins associated with PI3k signaling. At short incubation times, cells do not appear to influence each other, regardless of cell separation. For 6 h incubation times, the cells exert an inhibiting influence on each other at short separations and a predominately activating influence at large separation. Protein-specific cell-cell interaction functions are extracted, and by assuming pairwise additivity of those interactions, the functions are shown to correctly predict the results from three-cell experiments carried out under the identical conditions.

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.

Significance Reduced oxygen supply—hypoxia—is a near-universal feature of solid tumors that can alter how tumors respond to therapies. We investigated the transition from normoxia to hypoxia in model brain cancer systems, using single-cell proteomics and data analysis tools based on physicochemical concepts. This approach permits the simplification of otherwise complex biology. We find a hypoxia-induced switch within a mammalian target of rapamycin (mTOR) signaling network. At the switching point, mTOR is predicted, and then shown by experiment, to be unresponsive to inhibition. These results may help explain the undistinguished performance of mTOR inhibitors in certain clinical trials.

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. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network.

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. These methods can provide a direct correlation of morphological, functional and molecular signatures at the single-cell level. We also provide examples of how those platforms are being applied to both fundamental biology and clinical studies, focusing on immune-system monitoring and phosphoprotein signaling networks in cancer.

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 0.65, 0.57, 0.49, 0.41, and 0.33 kV/cm were simultaneously produced in a single chip when the voltage of 1.3 kV was applied. We transferred enhanced green fluorescent protein genes (pEGFP) into HEK-293 and CHO cells, which were cultured within the microchannels. The feasibility of our device was demonstrated because it was able to produce five different transfection rates and survival rates at different electric fields produced in a single microchip. This system is expected to optimize the experimental conditions in gene transfection research more easily and faster than conventional electroporation methods.

ChemPhysChemVolume 11, Issue 14 p. 3063-3069 CommunicationFree Access Chemistries for Patterning Robust DNA MicroBarcodes Enable Multiplex Assays of Cytoplasm Proteins from Single Cancer Cells Young Shik Shin, Young Shik Shin Division of Chemistry and Chemical Engineering, Nanosystems Biology Cancer Center, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA), Fax: (+1)626-395-2355 Division of Engineering and Applied Science, Bioengineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA) These authors contributed equally to this work.Search for more papers by this authorHabib Ahmad, Habib Ahmad Division of Chemistry and Chemical Engineering, Nanosystems Biology Cancer Center, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA), Fax: (+1)626-395-2355 These authors contributed equally to this work.Search for more papers by this authorDr. Qihui Shi, Dr. Qihui Shi Division of Chemistry and Chemical Engineering, Nanosystems Biology Cancer Center, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA), Fax: (+1)626-395-2355 These authors contributed equally to this work.Search for more papers by this authorDr. Hyungjun Kim, Dr. Hyungjun Kim Graduate School of EEWS, Korea Advanced Institute of Science Technology, Daejeon, 305-701(Republic of Korea) Materials and Process Simulation Center, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA)Search for more papers by this authorDr. Tod A. Pascal, Dr. Tod A. Pascal Graduate School of EEWS, Korea Advanced Institute of Science Technology, Daejeon, 305-701(Republic of Korea) Materials and Process Simulation Center, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA)Search for more papers by this authorProf. Rong Fan, Prof. Rong Fan Department of Biomedical Engineering, Yale University, PO Box 208260, New Haven, CT 06520 (USA)Search for more papers by this authorProf. William A. Goddard III, Prof. William A. Goddard III Graduate School of EEWS, Korea Advanced Institute of Science Technology, Daejeon, 305-701(Republic of Korea) Materials and Process Simulation Center, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA)Search for more papers by this authorProf. James R. Heath, Prof. James R. Heath heath@caltech.edu Division of Chemistry and Chemical Engineering, Nanosystems Biology Cancer Center, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA), Fax: (+1)626-395-2355Search for more papers by this author Young Shik Shin, Young Shik Shin Division of Chemistry and Chemical Engineering, Nanosystems Biology Cancer Center, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA), Fax: (+1)626-395-2355 Division of Engineering and Applied Science, Bioengineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA) These authors contributed equally to this work.Search for more papers by this authorHabib Ahmad, Habib Ahmad Division of Chemistry and Chemical Engineering, Nanosystems Biology Cancer Center, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA), Fax: (+1)626-395-2355 These authors contributed equally to this work.Search for more papers by this authorDr. Qihui Shi, Dr. Qihui Shi Division of Chemistry and Chemical Engineering, Nanosystems Biology Cancer Center, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA), Fax: (+1)626-395-2355 These authors contributed equally to this work.Search for more papers by this authorDr. Hyungjun Kim, Dr. Hyungjun Kim Graduate School of EEWS, Korea Advanced Institute of Science Technology, Daejeon, 305-701(Republic of Korea) Materials and Process Simulation Center, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA)Search for more papers by this authorDr. Tod A. Pascal, Dr. Tod A. Pascal Graduate School of EEWS, Korea Advanced Institute of Science Technology, Daejeon, 305-701(Republic of Korea) Materials and Process Simulation Center, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA)Search for more papers by this authorProf. Rong Fan, Prof. Rong Fan Department of Biomedical Engineering, Yale University, PO Box 208260, New Haven, CT 06520 (USA)Search for more papers by this authorProf. William A. Goddard III, Prof. William A. Goddard III Graduate School of EEWS, Korea Advanced Institute of Science Technology, Daejeon, 305-701(Republic of Korea) Materials and Process Simulation Center, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA)Search for more papers by this authorProf. James R. Heath, Prof. James R. Heath heath@caltech.edu Division of Chemistry and Chemical Engineering, Nanosystems Biology Cancer Center, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 (USA), Fax: (+1)626-395-2355Search for more papers by this author First published: 16 August 2010 https://doi.org/10.1002/cphc.201000528Citations: 46 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract The optimization of chemistries to enable the patterning of miniaturized DNA barcodes using microfluidics flow channels is described (see picture). Experiment and theory reveal that solvent mixtures in which counterions are strongly associated with the negatively charged DNA oligomers may be harnessed to produce high quality, high density DNA microarray patterns over a large area. The demand for parallel, multiplex analysis of protein biomarkers from ever smaller biospecimens is an increasing trend for both fundamental biology and clinical diagnostics.1–3 The most highly multiplex protein assays rely on spatially encoded antibody microarrays,4–6 and small biospecimens samples are now routinely manipulated using microfluidics approaches. The integration of antibody microarray techniques with microfluidics chips has only been explored relatively recently. One challenge arises from the relative instability of antibodies to microfluidics fabrication conditions. In recent years, several groups have devised methods to transform standard DNA microarrays in situ into protein microarrays and cell-capture platforms.7–13 These approaches capitalize on the chemical robustness of DNA oligomers, and the reliable assembly of DNA-labeled structures via complementary hybridization. Recently, Fan et al. utilized a microfluidics-based flow patterning technique to generate DNA barcode-type arrays at 10× higher density than standard, spotted microarrays.14 The DNA barcodes were converted into antibody arrays using the DNA-encoded antibody library (DEAL) technique, and then applied towards the measurement of a highly multiplex panel of proteins from a pinprick of whole blood. A second challenge involves scaling such miniaturized DNA microarrays so that a large surface area can be encoded. This problem is non-trivial, as it involves identifying chemistries for patterning 10−5 m wide, 1 m long strips of biomolecules with a uniformity that permits those patterns to be utilized in hundreds to thousands of quantitative protein assays per chip. Herein, we explore the surface chemistry associated with microfluidics-based flow patterning of DNA barcodes, with an eye towards producing highly reproducible and robust barcodes. We then apply the optimized chemistry towards assaying a panel of cytoplasmic proteins from single cells. We explore three different flow patterning surface chemistries: two rely upon the electrostatic adsorption of DNA onto a poly-L-lysine (PLL) surface, and the third utilizes flow patterning of dendrimers onto aminated glass substrates, followed by covalent attachment of DNA oligomers onto the dendrimer scaffolds. For the electrostatic adsorption cases, we investigate, using both theory and experiment, the role that counterions play in flow patterning within the confined dimensions of a microfluidic channel, and we find that solvent mixtures which associate counterions more strongly to the negatively charged DNA oligomers yield more reproducible and robust barcodes. We then demonstrate the utility of the best flow patterning chemistry by combining it with DEAL to construct antibody barcodes for quantitatively assaying a panel of phosphorylated proteins, associated with oncogenic pathways, from single cells that are representative of the brain cancer glioblastoma multiforme (GBM). The microfluidics flow patterning chip is comprised of a patterned polydimethylsiloxane (PDMS) layer adhered to an aminated or PLL-coated glass substrate that provides the base surface for the microchannels. The microchannels are long (about 55 cm), meandering channels that span ca. 0.85 cm2 of our substrate, and are used to pattern a DNA barcode over most of the glass surface (Figure 1 b). After the flow patterning is completed, the PDMS layer is replaced with a second micropatterned PDMS layer that is designed to support a biological assay, such as the previously reported blood proteomics chip,14 or the single-cell proteomics chips utilized herein. For the microfluidic patterning method to be useful, it must generate a DNA barcode that exhibits high and uniform DNA loading over the entire substrate. We evaluated the patterning chemistries illustrated in Figure 1 a, Schemes 1–3. Schemes 1 and 2 are drawn from the conventional protocol for pin-spotted microarrays—a solution containing the DNA is introduced, the solvent is evaporated, and subsequent thermal or UV treatment is employed to cross-link the deposited DNA to the substrate. In Scheme 1 ssDNA oligomers dissolved in phosphate-buffered saline (PBS) are utilized, whereas in Scheme 2 ssDNAs in a 1:1 mixture of 1×PBS and dimethyl sulfoxide (DMSO) are employed. DMSO is used in conventional microarray preparation to improve feature consistency by reducing the rate of solvent evaporation and by denaturing the DNA15 although, as described below, its role in this process is different. In Scheme 3 a covalent immobilization method based upon a dendrimer scaffold is utilized.16 Poly(amidoamine) (PAMAM) dendrimers (generation 4.5, carboxylate surface) have previously shown promise as DNA and protein microarray substrates. Dendrimers do not form entangled chains17 and because harsh crosslinking procedures are avoided, dendrimer-immobilized DNA retains high accessibility and activity in microarray applications. Moreover, the highly branched structure of the dendrimers provides a high density of reactive sites for surface attachment and for DNA coupling, thus leading to a high overall binding capacity. For all cases, a high level of DNA loading has been shown to decrease non-specific binding when compared to standard microarray substrates.11, 18–20 Figure 1Open in figure viewerPowerPoint a) Surface treatment schemes. b) Design of the DNA patterning device (top) and fluorescence image of DNAs filled into the channel (still in solution). Outer five channels are filled with DNAs in 1:1 mixture of PBS and water (Scheme 1 ). The five inner are filled with DNA in a 1:1 mixture of PBS and DMSO (Scheme 2 ). Three channels in between are left empty for visualization. c) Fluorescence images of patterned DNAs by three schemes. Figure 1 b (top) shows the PDMS chip design used for barcode patterning. Thirteen discrete channels (for a thirteen-element barcode) allow for a multiplex microarray. We loaded five adjacent channels according to Scheme 1, skipped three channels, and then loaded the remaining five channels according to Scheme 2. The use of fluorescently-tagged DNA permitted measurements of the DNA distribution within each individual channel immediately after introducing the solutions. Figure 1 b demonstrates a clear difference in aqueous DNA distribution across the chip: DNA loaded according to Scheme 1 (outer five channels) is notably lower in concentration near the middle of the chip (Figure 1 b, Region 2) and is barely detectable near the channel exit (Figure 1 b, Region 1). Conversely, DNA loaded according to Scheme 2 (inner five channels) presents an even, consistent distribution across the entire chip. Notably, Scheme 1 yields a relatively higher fluorescence intensity at the input side of the chip. These results clearly indicate that, for Scheme 1, the ssDNA oligomers are accumulating upstream during the early stages of flow, and so are depleted from the advancing solution by the time it reaches mid-chip. The actual patterning of the glass substrate occurs when solvent is evaporated (Figure S2, Supportiing Information). Indeed, the final patterning results after solvent evaporation and cross-linking (Figure 1 c, top) reflect the trend established by the aqueous fluorescence images; Scheme 2 produces uniform DNA barcodes across the substrate, while Scheme 1 does not. Line profiles corresponding to Figure 1 c can be found in Figure S1 (Supporting Information). In order to understand the difference in patterning uniformity between Schemes 1 and 2, we considered the electrostatic environment for each case. As depicted in Figure 2 a, the PDMS side walls carry a slightly negative zeta potential, whereas the PLL surface has a strong positive zeta potential.21 When the ssDNA solution in Scheme 1 is introduced to the channel, ssDNA near the PLL matrix is electrostatically immobilized, thereby generating a concentration gradient.22 As the solution flows towards the channel exit, the ssDNA oligomers are continually depleted via deposition onto the PLL surface. Figure 2 b shows the results from a rough simulation designed to capture the mean concentration of aqueous ssDNA as the solution traverses a channel. The simulation implies that the effect of electrostatic adsorption proves dominant even at high DNA concentrations, a result that agrees well with the observed behavior for Scheme 1 in Figure 1 b. A detailed description of the model and assumptions employed can be found in the Supporting Information. We tested this model via the strong negative charging of all four channel surfaces via O2 plasma treatment. Consistent with the model, both Schemes 1 and 2 exhibited equivalently uniform distribution of fluorescence intensity across the chip (Figure S3 b, Supporting Information). We note that lack of the positive charges on the bottom surface failed to hold DNAs during the drying procedure and that the plasma treatment induces the irreversible bonding of PDMS and glass, which limits further use beyond this experimental test. Figure 2Open in figure viewerPowerPoint Electrostatic adsorption of DNAs on PLL surface and DMSO effect. a) The filling step. b) Simulation result of electrostatic adsorption of DNAs to PLL surface. c) Molecular simulation of the DMSO effect: the radial distribution function of P atom of the phosphate group and the sodium ions. The presence of DMSO pumps sodium ions from the 2nd shell to the 1st shell (arrow). d) Schematics for DMSO effect. Green circles represent sodium ions. The results from Schemes 1 and 2 imply that DMSO alleviates the electrostatic adsorption effect. In order to understand this more fully, we performed molecular dynamics (MD) simulations of DNA in PBS and PBS/DMSO solutions; 3 ns of NPT [NPT is a simulation in which number of moles (N), pressure (P) and temperature (T) are held constant]. The MD simulations were performed with the last 1 ns trajectory used for analysis. We examined the radial distribution function of phosphorous atoms in the DNA backbone with respect to various elements of the surrounding solvent. For example, the radial distribution function of P and the O atom of a water molecule is virtually unperturbed by the addition of DMSO (Figure S4, Supporting Information). Consequently, it is unsurprising that the radial distribution function of P and the S atom of DMSO (Figure 2 c, black solid line) reveals that DMSO is not forming a solvation structure with the DNA backbone. However, Figure 2 c demonstrates a clear interaction between P and Na+ ions, which delineates into two well-defined shell structures: the first is located at r<4.3 Å while the second is located at 4.3 Å<r<6.6 Å. These are similar to the locations of the first and the second water solvation structures. By integrating the radial distribution functions, we determined the number of molecules per phosphate in the first and second shells for both PBS and PBS/DMSO solutions. Although the number H2O molecules per shell is virtually independent of DMSO, DMSO does significantly increase the number of Na+ ions in the first shell (from 0.14 to 0.24), and it decreases the number of Na+ ions in the second shell (from 0.61 to 0.34). Conversely, the number of DMSO molecules is almost zero in the first shell (0.01) but becomes significant in the second shell (0.20). Thus, we conclude that DMSO, with a lower dielectric constant relative to water (47.2 vs 80), destabilizes the solvation energy of Na+ in the second shell. This thermodynamic change prompts the sodium ions to move to the first shell where they are stabilized by electrostatic interactions with the negatively charged phosphate groups. The increased number of sodium ions near the DNA backbone screens the negative charges of phosphate groups more efficiently, thereby reducing electrostatic interactions of the DNA with the PLL surface, resulting in uniform DNA distribution throughout the channels. Although the addition of DMSO to DNA patterning solutions yields the same ultimate effect for both traditional spotted arrays and microfluidics-patterned barcodes, the underlying mechanisms are completely different. We conclude that Scheme 2 is intrinsically superior relative to Scheme 1. We now turn towards analyzing Scheme 3, and comparing it against Scheme 2. For this scheme, the PAMAM dendrimers are first covalently attached to the aminated glass surface, and then (aminated) ssDNA oligomers are covalently attached to the dendrimers. The lack of a solvent evaporation step makes Scheme 3 significantly more rapid than Scheme 2. We flowed activated PAMAM dendrimers, followed by aminated ssDNA, through ten microfluidic channels (Figure 1 b). Note that the aqueous DNA distribution is expected to be uniform because the substrate surface is comprised of charge-neutral N-hydroxysuccinimide (NHS)-modified carboxylates which minimize electrostatic interactions. The resulting DNA microarray was assayed for uniformity with complementary DNAs labeled with Cy3-fluorophores. Visual analysis indicates good uniformity across the chip (Figure 1 c, bottom). In order to quantify the patterning quality for all three schemes, we obtained signal intensities for each channel at sixteen locations within the patterning region and calculated the coefficient of variation (CV). The CV is defined as the standard deviation divided by the mean and expressed as a percentage. CVs for Schemes 1, 2, and 3 registered 69.8 %, 10.5 %, and 10.9 %, respectively. Thus, we conclude that Schemes 2 and 3 offer consistent DNA loading across the entire substrate. Having established that Schemes 2 and 3 produce consistent, large-scale DNA barcodes, we then extended our analysis of array consistency to protein measurements. We previously demonstrated that, when using the DEAL platform for multiplex protein sensing in microfluidics channels, the sensitivities of the assays directly correlate with the amount of immobilized DNA,14 up to the point where the DNA coverage is saturated. We performed multiple protein assays along the length of our DNA stripes to ensure that the results described above would translate into stable and sensitive barcodes for protein sensing. All protein assays were performed in microfluidic channels which were oriented perpendicular to the patterned barcodes (five channels for Scheme 2 and four channels for Scheme 3). This allowed us to test distal microarray repeats with a single small analyte volume. For barcodes prepared using Scheme 2, we utilized the DEAL technique to convert them into antibody barcodes designed to assay the following proteins: phosphorylated (phospho)-steroid receptor coactivator (Src), phospho-mammalian target of rapamycin (mTOR), phospho-p70 S6 kinase (S6K), phospho-glycogen synthase kinase (GSK)-3α/β, phospho-p38α, phospho-extracellular signal-regulated kinase (ERK), and total epidermal growth factor receptor (EGFR) at 10 ng mL−1 and 1 ng mL−1 concentrations. This panel samples key nodes of the phosphoinositide 3-kinase (PI3K) signaling pathway within GBM, and are used below for single-cell assays.23 For barcodes prepared using Scheme 3, we similarly converted the DNA barcodes into antibody barcodes designed to detect three proteins [interferon (INF)-γ, tumor necrosis factor (TNF)α, and interleukin (IL)-2] at 100 ng mL−1 and 10 ng mL−1. All the DNAs used were pre-validated for the orthogonality in order to avoid cross-hybridization and the sequences can be found in the Supporting Information, Table 1. The detection scheme is similar to a sandwich immunoassay. Captured proteins from primary antibodies were fluorescently visualized by biotin-labeled secondary antibodies and Cy5-labeled streptavidin. For both cases, data averaged from multiple DNA repeats across the chip yielded CVs that were commensurate with those of the underlying DNA barcodes (from 10 ng mL−1 concentration, 7 % for scheme 2 and 17 % for Scheme 3, respectively). Figure 3 shows line profiles of the signal intensities along with the raw data, and demonstrate a better uniformity for barcodes prepared according to Scheme 2. While we found that Scheme 3 could produce barcodes that were close in quality to those of Scheme 2, the absolute (chip-to-chip) consistency of Scheme 3 is hard to guarantee due to its use of the unstable coupling reagents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and NHS.24 Moreover, although Scheme 3 is faster, the detailed procedure itself is more labor-intensive. Scheme 2 can potentially be automated. Thus, we chose Scheme 2 as the preferred barcode patterning method. With Scheme 2 , over 90 % of the patterned slides showed good quality for the test. Figure 3Open in figure viewerPowerPoint Contrast-enhanced raw data extracted from multi-protein calibration experiments performed on a substrate prepared according to a) Scheme 2 and b) Scheme 3 . Each red bar represents a unique protein measurement, and is clustered with up to ten additional proteins (for Scheme 2 ). The clusters become symmetrical due to the winding nature of the barcode pattern, so that each cluster actually contains two measurements of each protein. Clustering is less evident in (b) because lower-density barcode pattern was employed. Recombinant proteins were analyzed across five discrete channels per concentration for (a) and four discrete channels per concentration for (b); quantitative data for statistical analysis was extracted from all the repeats in each of the channels. By utilizing identical DEAL cocktails followed by identical standard protein cocktails, the reproducibility was also checked. The identical signal patterns within individual channels and between channels of similar concentrations demonstrate the good uniformity and quality of DNA barcodes. Signal intensity profiles sampled from one analysis channel per concentration are quantified in white. Scale bar: 2 mm. We validated the use of the antibody barcodes by applying them towards the multiplex assay of cytoplasmic proteins from single cells. There is a significant body of evidence that demonstrates that genetically identical cells can exhibit significant functional heterogeneity—behavior that cannot be captured by proteomics techniques that average data across a population.25 We therefore designed a highly parallel microfluidic device capable of isolating single/few numbers of cells in chambers with a full complement of antibody barcodes designed to detect intracellular proteins (Figure S5, Supporting Information). Figure 4 a shows a schematic of the device and the DEAL-based protein detection scheme. The small chamber size keeps the finite number of protein molecules concentrated, thereby enhancing sensitivity. Assaying such a panel of proteins would not be possible without a high density antibody array, such as the barcodes utilized herein, for the following reasons. First, all the barcodes should fit into such a small chamber for multiplexing. Second, since data averaging in such a spatially-constrained scheme is impractical, it is critical to have consistent DNA loading across the microrarray if data comparisons are to be meaningful. Figure 4Open in figure viewerPowerPoint a) Schematic representation of the single-cell, intracellular protein analysis device. Single or few cells are incubated in an isolated chamber under varying stimuli. Intracellular proteins are assayed by introducing a pre-aliquoted lysis buffer, whereupon the released proteins bind to the DEAL (DNA-labeled antibody) barcode within the chamber. V1: valve for lysis buffer control, V2: valve for isolated chamber formation, and R1: DNA barcode array converted into DEAL antibody array. b), c) Contrast-enhanced images of developed barcode assays highlight the benefits of using Scheme 2 (b) versus Scheme 1 (c). Protein names listed in red font correspond to those which were detected using Scheme 2 barcodes. d) Representative fluorescence intensity profile from the single-cell lysate of (b). We chose the U87 GBM cell line as a model system for our platform. GBM is the most common malignant brain tumor found in adults, and is the most lethal of all cancers. As the name implies, GBM exhibits extensive biological variability and heterogeneous clinical behavior.26 EGFR is an important GBM oncogene and therapeutic target.27 Thus, we assayed for eleven intracellular proteins associated with the EGFR-activated PI3 K signaling pathway. We provide representative sets of data for protein detection from the lysate of one to five cells (Figures 4 b and c). Eight proteins were detected from single-cell lysate and up to nine proteins were detected from five cells when using barcodes patterned by Scheme 2 (Figures 4 b, d), whereas only one protein could be detected from barcodes prepared by Scheme 1 (Figure 4 c). All the separate protein assays were screened for cross-reactivity (Figure S6), and, for the cases where recombinant proteins were available, quantitation curves for each protein assay were measured (Figure S7). More detailed statistical analysis of these cells, as well as genetic variants thereof, is currently being investigated. We identified a protocol for generating high-quality, high-density DNA barcode patterns by comparing three microfluidics-based patterning schemes. We find, through both experiment and theory, that the electrostatic attractions between positively-charged PLL substrates and the negatively-charged DNA backbone induces significant non-uniformity in the patterning process, but that those electrostatic interactions may be mediated by adding DMSO to the solution, resulting in uniform and highly reproducible barcodes patterned using ∼55 cm long channels that template barcodes across an entire 2.5 cm wide glass slide. Dendrimer-based covalent immobilization also yields good ultimate uniformity, but is hampered by a relatively unstable chemistry that limits run-to-run reproducibility. DNA barcodes were coupled with the DEAL technique to generate antibody barcodes, and then integrated into specifically designed microfluidic chips for assaying cytoplasm proteins from single and few lysed U87 model cancer cells. Successful detection of a panel of such proteins represents the potential of our platform to be applied to various biological and, perhaps, clinical applications. Experimental Section Microfluidic Chip Fabrication for DNA Patterning: Microfluidic-patterning PDMS chips were fabricated by soft lithography. The master mold was prepared using either a negative photoresist, SU8 2010, with photolithography or an etched silicon mold generated by a deep reactive ion etching (DRIE) process. The mold has long meandering channels with a 20×20 μm cross section. The distance from channel to channel is also 20 μm, which generates 10× higher density than standard, spotted microarrays. Sylgard PDMS (Corning) prepolymer and curing agent were mixed in a 10:1 ratio (w/w), poured onto the mold, and cured (80°, 1 hour). The cured PDMS slab was released from the mold, inlet/outlet holes were punched, and the device was bonded onto a PLL coated (C40–5257 M20, Thermo scientific) or aminated glass slide (48382–220, VWR) to form enclosed channels. The number of microfluidic channels determines the size of the microarray; 13 parallel microchannels were used in this study. Patterning of DNA Barcode Arrays: For the DNA filling test, a 30-mer DNA oligomer labeled with Cy3 fluorescence tag on the 5' end (5′-/Cy3/-AAA AAA AAA ATA CGG ACT TAG CTC CAG GAT-3′) in a 1:1 mixture (v/v) of 1×PBS buffer and DMSO or a 1:1 mixture (v/v) of 1×PBS buffer and deionized (DI) water was used. The final DNA concentration was 2.5 μM. DNA solution

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 due to the heterogeneous reaction with O 3 , field induced droplet ionization (FIDI) mass spectrometry is utilized.FIDI is a soft ionization method in which ions are extracted from the surface of microlitervolume droplets.We report the structurally specific oxidative changes of SP-B 1-25 (a shortened version of human surfactant protein B) at the air-liquid interface.We also present studies of the interfacial oxidation of SP-B 1-25 in a non-ionizable 1-palmitoyl-2-oleoyl-sn-glycerol (POG) surfactant layer as a model PS system, where the 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.Compared to the nearly complete homogeneous oxidation of SP-B 1-25 , only a subset of the amino acids known to react with ozone is oxidized by direct ozonolysis in the hydrophobic interfacial environment, both with and without the lipid monolayer.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 when subject to oxidative stress.

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.

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. Our results demonstrate unique characteristics of the heterogeneous reactions at the air−liquid interface. We observe the hydroxyhydroperoxide and the secondary ozonide as major products of POPG ozonolysis in the FIDI-MS spectra. These products are metastable and difficult to observe in the bulk phase, using standard electrospray ionization (ESI) for mass spectrometric analysis. We also present studies of the heterogeneous ozonolysis of a mixture of saturated and unsaturated phospholipids at the air−liquid interface. A mixture of the saturated phospholipid 1,2-dipalmitoyl-sn-phosphatidylglycerol (DPPG) and unsaturated POPG is investigated in negative ion mode using FIDI-MS while a mixture of 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) and 1-stearoyl-2-oleoyl-sn-phosphatidylcholine (SOPC) surfactant is studied in positive ion mode. In both cases FIDI-MS shows the saturated and unsaturated pulmonary surfactants form a mixed interfacial layer. Only the unsaturated phospholipid reacts with ozone, forming products that are more hydrophilic than the saturated phospholipid. With extensive ozonolysis only the saturated phospholipid remains at the droplet surface. Combining these experimental observations with the results of computational analysis provides an improved understanding of the interfacial structure and chemistry of a surfactant layer system when subject to oxidative stress.

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. The microelectroporation chip has a microchannel with a height of 20 μm and a length of 2 cm. Owing to the transparency of PDMS, we could in situ observe the uptake process of propidium iodide (PI) into SK-OV-3 cells, which shows promise in visualization of gene delivery in living cells. We also noticed the geometric effect on the degree of electroporation in microchannels with diverse channel width. This experimental result shows that the geometry can be another parameter to be considered for the electroporation when it is performed in microchannels with an exponential decaying pulse generator. Cell culturing is possible within the microelectroporation chip, and we also successfully transfected SK-OV-3 cells with enhanced green fluorescent protein genes, which demonstrates the feasibility of the microelectroporation chip in genetic manipulation.

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.

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.

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.

Al2O3 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 Al2O3 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 Al2O3, especially in nanometer-scale electronic device settings. In this work, electron transport via Al2O3 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. First, it is confirmed that the transmission function, T(E), decreases as a function of energy in (E – EF) < 0 regime, which is an intuitively expected trend. On the other hand, in the (E – EF) > 0 regime, Al2O3(100) and Al2O3(012) show their own characteristic behaviors of T(E), presenting that major peaks are shifted toward lower energy levels under a finite bias voltage. Second, the overall conductance decay rates under zero bias are similar regardless of the crystallographic orientation, so that the contact interface seemingly has only a minor contribution to the overall conductance. A noteworthy feature at the finite bias condition is that the electrical current drastically increases as a function of bias potential (>0.7 V) in Al2O3(012)-based junction compared with the Al2O3(100) counterpart. It is elucidated that such a difference is due to the well-developed eigenchannels for electron transport in the Al2O3(012)-based junction. Therefore, it is evidently demonstrated that at finite bias condition, the contact interface plays a key role in determining insulating properties of Al2O3–Pt junctions.

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 ( 18 F-FDG). We report on an integrated microfluidics-chip/beta particle imaging system for in vitro 18 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. Further, we find a weak positive correlation between cell size and rate of glycolysis.

The degeneration of two classes of deep beam elements is conducted, one (DB6) based on the conventional Timoshenko beam assumptions and the other (DB7) based on the assumed cubic order longitudinal displacement profile. While an adjustable shear correction factor is required for the DB6 element to compensate for the unrealistic distribution of a shear strain across the beam depth, the DB7 element assumes the more realistic quadratic profile of shear strain at the outset. With the plane‐stress continuum solution serving as reference in static and free‐vibration analyses, solutions obtained by these two element models are compared with the analytical Timoshenko solution, the analytical thin beam solution and several available solutions of existing beam elements. The result indicates that the performance of the higher order beam element DB7 is seen to be more versatile than other models previously developed by various investigators. Also, superior accuracy of the results is evident in both analyses over a wide range of the beam aspect ratios.

We investigate the transport of electrons in a ferrocene aqueous solution nanoconfined between two Pt electrodes using density functional theory and a nonequilibrium Green function method. The system consists of three characteristic phases: metal electrodes, the electrode–solution interface, and the nanoconfined solution phase. By performing the geometry optimization of such systems, it is found that the molecular configuration of water molecules at the Pt surface is adjusted due to the Pt–water interaction, and the charges at the Pt surface are redistributed. Next, by applying the external bias potential via the effective screening medium method during ab initio molecular dynamics simulations, it is revealed that water molecules are packed at the Pt–water interfacial region, and the electrostatic potential profile in the water phase is significantly changed due to aligned water layers. From the analysis of the electron transport characteristics, it is discovered that the ferrocene molecule generates a strong transmission function near the Fermi level and high electron density of states spatially connecting both electrodes through the water phase. These features explain the drastic enhancement in electrical current–voltage characteristics. Therefore, it is concluded that ferrocene enhances electron transport through nanoconfined solutions, indicating that it can be used as a signaling molecule in nanoscale systems for electrochemical sensor applications.

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-1 with an excellent repeatability (±0.004). The desired pH microenvironment can be stably maintained for longer than 2 h within ±0.0012 pH. As a high-impact application of the platform technology, we propose a single-step immunoassay and demonstrate its utility in measuring C-reactive protein (CRP), a critical inflammatory marker in various conditions such as myocardial infarction and even SARS-Cov-2. Utilizing pH modulation technology along with pH-sensitive fluorescence dye simplifies the immunoassay process into a single-step, where a mixture of all of the reagents is incubated only for 1 h without any washing steps or the need to change solution. This simplified immunoassay process minimizes the hands-on time of the end-user and thus decreases technician-driven errors. Moreover, the absence of complicated liquid-handling hardware makes it more suitable and attractive for an ultracompact platform to ultimately be used in a point-of-care diagnostic assay.

This study deals with effects depending on skew angles in skewed-laminated composite materials in macroscopic point of view. Based on higher-order approximation of displacements, subparametric layer-wise finite elements are used to analyze skewed-laminated composite systems. The elements have higher-order shape functions derived from the Lobatto shape functions. The modes of the elements are classified into three groups such as vertex, side, and internal modes. The vertex modes have physical meaning, while side and internal modes with respect to the increase of order of the Lobatto shape functions do not have physical meaning but improve accuracy of analysis. Therefore, fixing mesh arrangement of present analysis, the quality of the analysis can be enhanced without re-meshing work. The approach based on p-version of finite element method is implemented with three-dimensional elasticity theory, while shape functions are developed by combination of one- and two-dimensional shape functions, not using three-dimensional shape functions. Using the accurate and practical proposed technique, macroscopic behavior of skewed-laminated composite materials is investigated.

A radiological observation was made of 466 cases of male rag-pickers, aged 15 to 58 years old, who live now in the seven camps scattered in the city. Among the 545 rag-pickers accommodated at the seven camps, 466 cases (85.9 % ) were surveyed. Of these cases 333 (71.8%) were in the 20-29 age group and 8cases(1.7%) above 45 years.The results obtained were as follows:1) The radiologicaI findings revealed that 56 cases corresponding to 12.0% of the total examines had variousforms of pulmonary tuberculosis, as classified by the extent of the disease, 60.7 % for minimal, 26.8 % moderately advanced, 12.5% Far advanced.2) The highest prevalence rate(19.3%) was seen among the dwellers in Young Dung Po camp and the lowest(3.7%) in the camp of Choong Koo.3) The Prevalence rate was less than 10 % among the youngers under 24 years of age, the rate, however, increased with the increment of age, showing 13.3% at thc age of 25, 22.7 % at 39, and 75.0 % at 45 respectively.4) The Prevalence rate also increased in proportion to the length of duration as rag- pickers.5) Of the total 56 cases of pulmonary tuberculosis there were 31 (55.4%) new cases, 83.9% of which were minimal tuberculosis, while the remaining 25(44.6%) were old cases, 24.0 % of which being Far advanced.

Abstract Background: 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. Methods: We investigated a clinically relevant model of acquired cancer drug resistance in glioblastoma (GBM). GBM39 is a human-derived primary GBM line that is maintained by serial transplantation in xenografts. GBM39 expresses high levels of the epidermal growth factor receptor (EGFR) variant(v)III oncogene, which sensitizes tumor cells to inhibitors of the mammalian target of rapamycin (mTOR). Therefore, we used this model to study acquired resistance to the mTOR kinase inhibitor CC214-2, which suppresses the proliferation of EGFRvIII expressing GBMs. We utilized a microfluidic device as a main tool that enables multiplex single cell proteomics. Relative to bulk assays, it can provide deeper insights into the signaling activity within a phosphoprotein signaling network, and how that activity is influenced by a drug. In addition to providing average protein levels, such assays also yield information on network signaling coordination that can be extracted from the variance for any given protein, as well as correlations between proteins. We determined the structure of the hyperactivated phosphoprotein signaling networks from statistical numbers of single cancer cells separated from the tumor for the untreated vehicle, during response to CC214-2, and after resistance to CC214-2. Results: We find evidence that the tumors can rapidly adapt (within 2-3 days) to specific targeted inhibitors. The details of how the cells adapt, which are uniquely resolved by the single cell assays, can suggest effective therapy combinations. For a GBM murine model, tumor cells rapidly adapted to mTOR inhibition by activating ERK/Src signaling, suggesting that mTOR and ERK/Src signaling provided two independent druggable pathways for that tumor. Consonant with this hypothesis, we devised three therapeutic combinations and showed them to be highly effective in causing sustained clinical remission in vivo. A retrospective analysis revealed that mTOR signaling was a major driver of tumor growth in the untreated tumor, while ERK/Src signaling promoted resistance to mTOR inhibition. An additional study of a low passage, patient derived GBM cell line was also shown to exhibit rapid adaptation to single-agent targeted therapy in a manner that suggested effective combination therapies for cell killing. Finally, the analysis was shown to be feasible on a freshly resected patient GBM tumor. Conclusions: The data and analysis presented here provide evidence that tumor cells can respond rapidly to a therapy and restores the growth characteristics temporarily disrupted by the therapy. This resistance mechanism is intrinsic; the same cells that respond to the therapy also adapt and develop resistance to it. For the GBM models explored here, a single cell proteomics analysis of the phosphoprotein signaling networks can resolve the independent signaling modes that drive tumor growth in both the untreated and drug resistant states. Such an analysis can be rapidly carried out using untreated tumor biopsies, and so may represent a new approach for guiding the selection of targeted combination therapies with low anticipated resistance. Citation Format: Young Shik Shin, Wei Wei, Beatrice Gini, Paul Mischel, James Heath. Single cell phosphoproteomics identifies adaptive network dynamics of mTOR inhibitor resistance and defines effective combination therapy in glioblastoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR09.

Abstract Single-cell functional proteomics assays can connect genomic information to biological function through the use of 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 phospho-protein signaling networks in cancer cells, or for capturing high-resolution snapshots of immune system function in cancer patients participating therapies that involve immune system engineering and manipulation. We discuss advances in single cell proteomics platforms, with an emphasis the types of data that can be obtained, and how analysis of that data can provide deep insights into either designing therapies, or understanding patient responses to therapies. I will provide illustrative examples of how microchip-based single cell functional proteomics platforms are being applied to both fundamental biology and clinical studies. Citation Format: James R. Heath, Wei Wei, Young Shik Shin, Min Xue, Jing Zhou, Alex Sutherland, Jing Yu. Nanotechnology and single cell proteomics as a diagnostic tool. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY23-02. doi:10.1158/1538-7445.AM2014-SY23-02

Abstract The technology boom of single-cell metabolomics and proteomics has led to exciting biological discoveries in the past decade. It has become widely acknowledged that resolving the intratumoral heterogeneity at single cell resolution can aid the understanding of cancer therapeutic response and progression, as well as facilitate the development of therapeutic interventions. Here we present an integrated assay that simultaneously quantifies the intracellular metabolites, glucose uptake, metabolic proteins and functional signaling proteins at the single-cell level. The measurement of metabolites is achieved through a series of competitive binding assays and is incorporated into the microfluidic-based single cell barcode chip platform. We demonstrate that this assay is capable of obtaining information from both protein and metabolic signaling pathways associated with tumor survival and progression, providing new insights into cancer heterogeneity and drug responsiveness. Citation Format: Min Xue, Wei Wei, Yapeng Su, Young Shik Shin, Jungwoo Kim, James R. Heath. Developing integrated single-cell metabolic/proteomic assays. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1995. doi:10.1158/1538-7445.AM2015-1995

The nature of civil infrastructure projects can be described as complex and large-scale systems that are dynamically associated with diverse organizations and participants. Large budgets, collaboration efforts, and IT strategies are involved in major projects to increase the efficiency and effectiveness of all site activities involved. Emerging IT-based computational environments are providing a feasible vision of ubiquitous computing for the improvement of productivity and efficiency. This paper introduces the serviceability criteria of wireless sensor network to investigate the practical and technical considerations. Among the criteria, reliability issue was chosen for experimental analysis to analyze the equivalent distance between receiver and transmitter. The performance measurement indicates that the degraded ratio compared with the line-of-sight condition increases in accordance with the thickness and dielectric constant of construction material.

This thesis describes technology platforms for various biological applications at nano- and microscale. The first platform is the silicon nanowire (SiNW) field-effect-transistor (FET)-based biosensor. SiNW FETs have unique features such as label-free, real-time, and electrical measurement, which will be demonstrated with DNA and protein sensing. We further demonstrate that using different surface chemistry can modulate the sensitivity and dynamic range of the sensor. Debye screening, one of the major bottlenecks of the technology, is shown to be circumvented by using electrostatically immobilized capture DNA for DNA sensing and a small synthetic capture agent, peptide, for protein sensing. A model for the detection of analyte by SiNW sensors is also developed and utilized to extract DNA binding kinetic parameters, which shows the potential of the platform as a more sensitive version of surface plasmon resonance (SPR). The second part of this thesis focuses on a more practical and easily expandable technology, the microfluidics-based platform, to perform a single-cell-based protein analysis. We develop a flow patterning technology to generate highly parallel DNA barcodes that can be further utilized as a handle to immobilize protein capture agents, such as antibodies. As a first step, a protocol to make high-quality DNA micro-barcodes with an excellent uniformity is introduced. The uniform DNA barcode patterns enable us to perform protein detection from single cells in a microfluidic device that spans the whole glass microscope slide. A data set from about thousand experiments can be collected from a single test with the developed microfluidic device, owing to the good quality of DNA barcodes and DNA Encoded Antibody Libraries (DEAL) technology. This platform further demonstrates that multi-parameter protein detection at the single-cell level presents cellular heterogeneity which leads to new findings in biology. A quantitative version of the Le Chatelier’s principle, as derived using information theory, is applied to analyze a large amount of data from this platform. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein–protein interaction network. Lastly, another application of microfluidics is demonstrated for studying interfacial chemistry on lung surfactant systems under oxidative stress, along with mass spectrometry (MS) and molecular dynamic (MD) simulation results. The findings from the MS and MD simulations provide mechanistic details for the reaction of ozone with unsaturated phospholipids, leading to possible damage of the pulmonary system by ROS or direct ozone exposure. These investigations focus on molecular transformations that occur as a result of oxidative stress. Such molecular transformations can have a strong influence on the physical properties of the pulmonary surfactant (PS) system (i.e., the surface tension and elasticity of the interface), and therefore understanding how chemical transformations influence such physical properties can provide key insights into how the PS system responds to environmental challenges. Thus, we also propose utilizing microbubbles as a model system for investigating the physical transformations of the PS system when exposed to environmental challenges. The chemical composition change, along with physical property change, is analyzed by altered bubble size and oscillatory behavior which can provide an improved understanding of the physics of a PS system when it is subjected to oxidative stress.

Abstract Pathophysiology of a complex disease such as cancer requires capability to analyze highly heterogeneous cellular events at single cell level, which can measure large numbers of molecular events from individual cells to be analyzed in statistically meaningful manner. Here we report on an approach that integrates microfluidic cell handling and in situ protein secretion profiling to assess the functional heterogeneity of single cells, with extensions to small cell colonies. We measured a dozen proteins secreted from cells for the most aggressive type of primary brain tumor, glioblastoma multiforme (GBM). We observed functional phenotypes in terms of secreted proteins with profound cellular heterogeneity but still in a statistically meaningful manner. The unique features that we confirmed from single cell analysis can present additional useful information to the conventional bulk analysis. Combining physical status of the system such as cell-cell distance and the protein secretion profiles enables to study tumor microenvironment. We further demonstrate the potential application of this platform to the clinic by analyzing solid tumor cells derived from a GBM patient. This platform is inexpensive, requires minute amounts of cells and yields large volume of molecular information, showing great potential for clinical assessment of cellular characteristics in human disease lesions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-141.

A new path-independent contour integral formula is presented to estimate the crack-tip integral parameter, J-value, for two-dimensional cracked elastic bodies which may quantify the severity of the crack-tip stress fields. The conventional J-integral method based on a line integral has been converted to an equivalent area or domain integral (EDI) by the divergence theorem. It is noted that the EDI method is very attractive because all the quantities necessary for computation of domain integrals are readily available in a finite element analysis. The details and its implementation are extended to the p-version FE model with hierarchic elements using integrals of Legendre polynomials. By decomposing the displacement field obtained from the p-version finite element analysis into symmetric and antisymmetric displacement fields with respect to the crack line, the Mode-I and Mode-II non-dimensional stress intensity factors can be determined by using the decomposition method. The example problems for validating the proposed techniques are centrally oblique cracked plates under tensile loading. The numerical results associated with the variation of oblique angles show very good agreement with the existing solutions. Also, the selective distribution of polynomial orders and the corner elements for automatic mesh generation are applied to improve the numerical solution in this paper. © 1998 John Wiley & Sons, Ltd.

Electroporation, is widely used method that can be directly applied to the field of gene therapy. However, very little is known about the basic mechanisms of DNA transfer and cell response to the electric pulse. As a more convenient and effective tool for the investigation on the mechanisms of electroporation and the enhancement of the functional efficiency, we developed a micro-electroporation chip with polydimethylsiloxane (PDMS). Owing to the transparency of PDMS, we could observe the process of Propidium Iodide (PI) uptake in real-time, which shows promise in visualization of gene activity in living cells. Furthermore, the design of the electroporation chip as a microchannel offers advantages in terms of efficiency in permeabilization of PI into SK-OV-3 cells. We also noticed the geometric effect on the degree of electroporation in microchannels with diverse channel width, which shows that the geometry can be another parameter to be considered for the electroporation when it is performed in microchannels.

The Graf stabilization has been introduced in treating lumbar spinal disorder associated with posterior instability. This study reviewed some problems of the Graf instrumentation as a soft stabilizer. The purpose of this study is to analyse the problems of the soft stabilization in spinal instability. We reviewed 145 cases which were operative treatment using the Graf instrument for lumbar spinal disorder associated with posterior instability at our department from May, 1991 to Dec, 1995. The mean follow up periods was 29 months ranging from 24 months to 6 years 8 months. Of the 145 cases, 22 cases were showed the problem. The diagnostic method were simple x-ray, flexion-extension lateral stress view and CT scan. Results were as follows: Adjacent segmental instability was 10 cases(6.9%), disc space narrowing was 8 cases(5.5%), screw loosening was 3 cases(2.1%) and breakage of the Graf band was 1 case(0.6%). The problems of the soft stabilization were adjacent segmental instability, disc space narrowing, screw loosening, and breakage of the Graf band. But the rate of adjacent segmental instability and disc space narrowing was lower than other lumbar spinal instrumentation.