Microgram-scale testing of reaction conditions in solution using nanoliter plugs in microfluidics with detection by MALDI-MS.
(41/1368)
This paper describes a microfluidic system to screen and optimize organic reaction conditions on a submicrogram scale. The system uses discrete droplets (plugs) as microreactors separated and transported by a continuous phase of a fluorinated carrier fluid. Previously, we demonstrated the use of a microfabricated PDMS plug-based microfluidic system to perform assays and crystallization experiments in aqueous solutions with optical detection. Here, we developed an approach that does not require microfabrication of microfluidic devices, is applicable to synthetic reactions in organic solvents, and uses detection by MALDI-MS. As a demonstration, conditions for selective deacetylation of ouabain hexaacetate were tested, and the optimum conditions for mono-, bis-, or trisdeacetylation have been identified. These conditions were validated by scale-up reactions and isolating these potentially neurotoxic products. Mono- and bisdeacetylated products are unstable intermediates in the deacetylation and were isolated for the first time. This system enables no-loss handling of submicroliter volumes containing a few micrograms of a compound of interest. It could become valuable for investigating or optimizing reactions of precious substrates (e.g., products of long synthetic sequences and natural products that can be isolated only in small quantities). (+info)
Refinement of optical map assemblies.
(42/1368)
MOTIVATION: Genomic mutations and variations provide insightful information about the functionality of sequence elements and their association with human diseases. Traditionally, variations are identified through analysis of short DNA sequences, usually shorter than 1000 bp per fragment. Optical maps provide both faster and more cost-efficient means for detecting such differences, because a single map can span over 1 million bp. Optical maps are assembled to cover the whole genome, and the accuracy of assembly is critical. RESULTS: We present a computationally efficient model-based method for improving quality of such assemblies. Our method provides very high accuracy even with moderate coverage (<20 x). We utilize a hidden Markov model to represent the consensus map and use the expectation-Maximization algorithm to drive the refinement process. We also provide quality scores to assess the quality of the finished map. AVAILABILITY: Code is available from www.cmb.usc.edu/people/valouev/ (+info)
Cell handling using microstructured membranes.
(43/1368)
Gentle and precise handling of cell suspensions is essential for scientific research and clinical diagnostic applications. Although different techniques for cell analysis at the micro-scale have been proposed, many still require that preliminary sample preparation steps be performed off the chip. Here we present a microstructured membrane as a new microfluidic design concept, enabling the implementation of common sample preparation procedures for suspensions of eukaryotic cells in lab-on-a-chip devices. We demonstrate the novel capabilities for sample preparation procedures by the implementation of metered sampling of nanoliter volumes of whole blood, concentration increase up to three orders of magnitude of sparse cell suspension, and circumferentially uniform, sequential exposure of cells to reagents. We implemented these functions by using microstructured membranes that are pneumatically actuated and allowed to reversibly decouple the flow of fluids and the displacement of eukaryotic cells in suspensions. Furthermore, by integrating multiple structures on the same membrane, complex sequential procedures are possible using a limited number of control steps. (+info)
Design of a side-view particle imaging velocimetry flow system for cell-substrate adhesion studies.
(44/1368)
Experimental models that mimic the flow conditions in microcapillaries have suggested that the local shear stresses and shear rates can mediate tumor cell and leukocyte arrest on the endothelium and subsequent sustained adhesion. However, further investigation has been limited by the lack of experimental models that allow quantitative measurement of the hydrodynamic environment over adherent cells. The purpose of this study was to develop a system capable of acquiring quantitative flow profiles over adherent cells. By combining the techniques of side-view imaging and particle image velocimetry (PIV), an in vitro model was constructed that is capable of obtaining quantitative flow data over cells adhering to the endothelium. The velocity over an adherent leukocyte was measured and the shear rate was calculated under low and high upstream wall shear. The microcapillary channel was modeled using computational fluid dynamics (CFD) and the calculated velocity profiles over cells under the low and high shear rates were compared to experimental results. The drag force applied to each cell by the fluid was then computed. This system provides a means for future study of the forces underlying adhesion by permitting characterization of the local hydrodynamic conditions over adherent cells. (+info)
Label-free quantitative proteomics using large peptide data sets generated by nanoflow liquid chromatography and mass spectrometry.
(45/1368)
We developed an integrated platform consisting of machinery and software modules that can apply vast amounts of data generated by nanoflow LC-MS to differential protein expression analyses. Unlabeled protein samples were completely digested with modified trypsin and separated by low speed (200 nl/min) one-dimensional HPLC. Mass spectra were obtained every 1 s by using the survey mode of a hybrid Q-TOF mass spectrometer and displayed in a two-dimensional plane with m/z values along the x axis, and retention time was displayed along the y axis. The time jitter of nano-LC was adjusted using newly developed software based on a dynamic programming algorithm. The comprehensiveness (60,000-160,000 peaks above the predetermined threshold detectable in 60-microg cell protein samples), reproducibility (average coefficient of variance of 0.35-0.39 and correlation coefficient of over 0.92 between duplicates), and accurate quantification with a wide dynamic range (over 10(3)) of our platform warrant its application to various types of experimental and translational proteomics. (+info)
Microfluidic techniques for single-cell protein expression analysis.
(46/1368)
BACKGROUND: The analysis of single cells obtained from needle aspirates of tumors is constrained by the need for processing. To this end, we investigated two microfluidic approaches to measure the expression of surface proteins in single cancer cells or in small populations (<50 cells). METHODS: One approach involved indirect fluorescence labeling of cell-surface proteins and channeling of cells in a microfluidic device past a fluorescence detector for signal quantification and analysis. A second approach channeled cells in a microfluidic device over detection zones coated with ligands to surface proteins and measured rates of passage and of retardation based on transient interactions between surface proteins and ligands. RESULTS: The fluorescence device detected expression of integrin alpha5 induced by basic fibroblast growth factor (FGF-2) treatment in MCF-7 cells and that of Her-2/neu in SK-BR-3 cells compared with controls. Experiments measuring passage retardation showed significant differences in passage rates between FGF-2-treated and untreated MCF-7 cells over reaction regions coated with fibronectin and antibody to integrin alpha5beta1 compared with control regions. Blocking peptides reversed the retardation, demonstrating specificity. CONCLUSIONS: Immunofluorescence detection in a microfluidic channel demonstrates the potential for assaying surface protein expression in a few individual cells and will permit the development of future iterations not requiring cell handling. The flow retardation device represents the first application of this technology for assessing cell-surface protein expression in cancer cells and may provide a way for analyzing expression profiles of single cells without preanalytical manipulation. (+info)
Transparent polymeric cell culture chip with integrated temperature control and uniform media perfusion.
(47/1368)
Modern microfabrication and microfluidic technologies offer new opportunities in the design and fabrication of miniaturized cell culture systems for online monitoring of living cells. We used laser micromachining and thermal bonding to fabricate an optically transparent, low-cost polymeric chip for long-term online cell culture observation under controlled conditions. The chip incorporated a microfluidic flow equalization system, assuring uniform perfusion of the cell culture media throughout the cell culture chamber. The integrated indium-tin-oxide heater and miniature temperature probe linked to an electronic feedback system created steady and spatially uniform thermal conditions with minimal interference to the optical transparency of the chip. The fluidic and thermal performance of the chip was verified by finite element modeling and by operation tests under fluctuating ambient temperature conditions. HeLa cells were cultured for up to 2 weeks within the cell culture chip and monitored using a time-lapse video recording microscopy setup. Cell attachment and spreading was observed during the first 10-20 h (lag phase). After approximately 20 h, cell growth gained exponential character with an estimated doubling time of about 32 h, which is identical to the observed doubling time of cells grown in standard cell culture flasks in a CO2 incubator. (+info)
Triggering and visualizing the aggregation and fusion of lipid membranes in microfluidic chambers.
(48/1368)
We present a method that makes it possible to trigger, observe, and quantify membrane aggregation and fusion of giant liposomes in microfluidic chambers. Using electroformation from spin-coated films of lipids on transparent indium tin oxide electrodes, we formed two-dimensional networks of closely packed, surface-attached giant liposomes. We investigated the effects of fusogenic agents by simply flowing these molecules into the chambers and analyzing the resulting shape changes of more than 100 liposomes in parallel. We used this setup to quantify membrane fusion by several well-studied mechanisms, including fusion triggered by Ca2+, polyethylene glycol, and biospecific tethering. Directly observing many liposomes simultaneously proved particularly useful for studying fusion events in the presence of low concentrations of fusogenic agents, when fusion was rare and probabilistic. We applied this microfluidic fusion assay to investigate a novel 30-mer peptide derived from a recently identified human receptor protein, B5, that is important for membrane fusion during the entry of herpes simplex virus into host cells. This peptide triggered fusion of liposomes at an approximately 6 times higher probability than control peptides and caused irreversible interactions between adjacent membranes; it was, however, less fusogenic than Ca2+ at comparable concentrations. Closely packed, surface-attached giant liposomes in microfluidic chambers offer a method to observe membrane aggregation and fusion in parallel without requiring the use of micromanipulators. This technique makes it possible to characterize rapidly novel fusogenic agents under well-defined conditions. (+info)