Femtomole mixer for microsecond kinetic studies of protein folding.
(33/884)
We have developed a microfluidic mixer for studying protein folding and other reactions with a mixing time of 8 mus and sample consumption of femtomoles. This device enables us to access conformational changes under conditions far from equilibrium and at previously inaccessible time scales. In this paper, we discuss the design and optimization of the mixer using modeling of convective diffusion phenomena and a characterization of the mixer performance using microparticle image velocimetry, dye quenching, and Forster resonance energy-transfer (FRET) measurements of single-stranded DNA. We also demonstrate the feasibility of measuring fast protein folding kinetics using FRET with acyl-CoA binding protein. (+info)
Models for excluded volume interaction between an unfolded protein and rigid macromolecular cosolutes: macromolecular crowding and protein stability revisited.
(34/884)
Statistical-thermodynamic models for the excluded volume interaction between an unfolded polypeptide chain and a hard sphere or hard rod cosolute are presented, permitting estimation of the free energy of transfer of a polypeptide chain with fixed radius of gyration from a dilute (ideal) solution to a solution containing volume fraction of either cosolute. Also presented is a general thermodynamic description of the equilibrium between a unique native state and a manifold of unfolded or partially unfolded states of a protein distinguished by their respective radii of gyration. Together with results of a Monte Carlo calculation of the distribution of radii of gyration of four different unfolded proteins published by Goldenberg in 2003, these models are used to estimate the effect of intermolecular excluded volume upon an experimentally measurable apparent two-state constant for equilibrium between native and nonnative conformations of each of the four proteins, and upon the experimentally measurable root mean-square radius of gyration of the unfolded protein. Model calculations predict that addition of inert cosolutes at volume fractions exceeding 0.1 stabilizes the native state relative to unfolded states by an amount that increases strongly with and with the size of the native protein relative to the size of inert cosolute, and results in significant compaction of the manifold of unfolded states. Predicted effects are in qualitative and/or semiquantitative accord with the results of several published experimental studies. (+info)
Particulate nature of blood determines macroscopic rheology: a 2-D lattice Boltzmann analysis.
(35/884)
Historically, predicting macroscopic blood flow characteristics such as viscosity has been an empirical process due to the difficulty in rigorously including the particulate nature of blood in a mathematical representation of blood rheology. Using a two-dimensional lattice Boltzmann approach, we have simulated the flow of red blood cells in a blood vessel to estimate flow resistance at various hematocrits and vessel diameters. By including white blood cells (WBCs) in the flow, we also calculate the increase in resistance due to white cell rolling and adhesion. The model considers the blood as a suspension of particles in plasma, accounting for cell-cell and cell-wall interactions to predict macroscopic blood rheology. The model is able to reproduce the Fahraeus-Lindqvist effect, i.e., the increase in relative apparent viscosity as tube size increases, and the Fahraeus effect, i.e., tube hematocrit is lower than discharge hematocrit. In addition, the model allows direct assessment of the effect of WBCs on blood flow in the microvasculature, reproducing the dramatic increases in flow resistance as WBCs enter short capillary segments. This powerful and flexible model can be used to predict blood flow properties in any vessel geometry and with any blood composition. (+info)
Accurate multiplex gene synthesis from programmable DNA microchips.
(36/884)
Testing the many hypotheses from genomics and systems biology experiments demands accurate and cost-effective gene and genome synthesis. Here we describe a microchip-based technology for multiplex gene synthesis. Pools of thousands of 'construction' oligonucleotides and tagged complementary 'selection' oligonucleotides are synthesized on photo-programmable microfluidic chips, released, amplified and selected by hybridization to reduce synthesis errors ninefold. A one-step polymerase assembly multiplexing reaction assembles these into multiple genes. This technology enabled us to synthesize all 21 genes that encode the proteins of the Escherichia coli 30S ribosomal subunit, and to optimize their translation efficiency in vitro through alteration of codon bias. This is a significant step towards the synthesis of ribosomes in vitro and should have utility for synthetic biology in general. (+info)
Wetting morphologies at microstructured surfaces.
(37/884)
The wetting of microstructured surfaces is studied both experimentally and theoretically. Even relatively simple surface topographies such as grooves with rectangular cross section exhibit a large variety of different wetting morphologies as observed by atomic force microscopy. This polymorphism arises from liquid wedge formation along the groove corners and from contact line pinning along the groove edges. A global morphology diagram is derived that depends only on two system parameters: (i) the aspect ratio of the groove geometry and (ii) The contact angle of the underlying substrate material. For microfluidics, the most interesting shape regimes involve extended liquid filaments, which can grow and shrink in length while their cross section stays essentially constant. Thus, any method by which one can vary the contact angle can be used to switch the length of the filament, as is demonstrated in the context of electrowetting. (+info)
Biomimetic autoseparation of leukocytes from whole blood in a microfluidic device.
(38/884)
Leukocytes comprise less than 1% of all blood cells. Enrichment of their number, starting from a sample of whole blood, is the required first step of many clinical and basic research assays. We created a microfluidic device that takes advantage of the intrinsic features of blood flow in the microcirculation, such as plasma skimming and leukocyte margination, to separate leukocytes directly from whole blood. It consists of a simple network of rectangular microchannels designed to enhance lateral migration of leukocytes and their subsequent extraction from the erythrocyte-depleted region near the sidewalls. A single pass through the device produces a 34-fold enrichment of the leukocyte-to-erythrocyte ratio. It operates on microliter samples of whole blood, provides positive, continuous flow selection of leukocytes, and requires neither preliminary labeling of cells nor input of energy (except for a small pressure gradient to support the flow of blood). This effortless, efficient, and inexpensive technology can be used as a lab-on-a-chip component for initial whole blood sample preparation. Its integration into microanalytical devices that require leukocyte enrichment will enable accelerated transition of these devices into the field for point-of-care clinical testing. (+info)
Rapid and simple quantification of bacterial cells by using a microfluidic device.
(39/884)
This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples. (+info)
Intravital microscopic observations of 15-microm microspheres lodging in the pulmonary microcirculation.
(40/884)
Vascular infusions of 15-microm-diameter microspheres are used to study pulmonary blood flow distribution. The sites of microsphere lodging and their effects on microvascular perfusion are debated but unknown. Using intravital microscopy of the subpleural surface of rat lungs, we directly observed deposition of fluorescent microspheres. In a pump-perfused lung model, approximately 0.5 million microspheres were infused over 30 s into the pulmonary artery of seven rats. Microsphere lodging was analyzed for the location in the microvasculature and the effect on local flow after lodging. On average, we observed 3.2 microspheres per 160 alveolar facets. The microspheres always entered the arterioles as singlets and lodged at the inlets to capillaries, either in alveolar corner vessels or small arterioles. In all cases, blood flow continued either around the microspheres or into the capillaries via adjacent pathways. We conclude that 15-microm-diameter microspheres, in doses in excess of those used in typical studies, have no significant impact on pulmonary capillary blood flow distribution. (+info)