Sachin Jambovane

Determination of kinetic parameters, Km and kcat, with a single experiment on a chip.

We have demonstrated a multistep enzyme reaction on a chip to determine the key kinetic parameters of enzyme reaction. We designed and fabricated a fully integrated microfluidic chip to have sample metering, mixing, and incubation functionalities. The chip generates a gradient of reagent concentrations in 11 parallel processors. We used beta-galactosidase and its substrate, resorufin-beta-D-galactopyranoside, as the model system of the enzyme reaction. With a single experiment on the chip, we determined the key parameters for the enzyme kinetics, K(m) and k(cat), and evaluated the effect of inhibitor concentrations on the reaction rates. This study provides a new tool for evaluating various effectors, such as inhibitors and cofactors, on the initial rate of an enzyme reaction, and it could be applied to a comprehensive bio/chemical reaction study.

Generating nonlinear concentration gradients in microfluidic devices for cell studies.

We describe a microfluidic device for generating nonlinear (exponential and sigmoidal) concentration gradients, coupled with a microwell array for cell storage and analysis. The device has two inputs for coflowing multiple aqueous solutions, a main coflow channel and an asymmetrical grid of fluidic channels that allows the two solutions to combine at intersection points without fully mixing. Due to this asymmetry and diffusion of the two species in the coflow channel, varying amounts of the two solutions enter each fluidic path. This induces exponential and sigmoidal concentration gradients at low and high flow rates, respectively, making the microfluidic device versatile. A key feature of this design is that it is space-saving, as it does not require multiplexing or a separate array of mixing channels. Furthermore, the gradient structure can be utilized in concert with cell experiments, to expose cells captured in microwells to various concentrations of soluble factors. We demonstrate the utility of this design to assess the viability of fibroblast cells in response to a range of hydrogen peroxide (H(2)O(2)) concentrations.

Log-scale dose response of inhibitors on a chip.

We demonstrate the accommodation of log-scale concentration gradients of inhibitors on a single microfluidic chip with a semidirect dilution capability of reagents for the determination of the half-inhibitory concentration or IC(50). The chip provides a unique tool for hosting a wide-range of concentration gradient for studies that require an equal distribution of measuring points on a logarithmic scale. Using Matrix metalloproteinase IX and three of its inhibitors, marimastat, batimastat, and CP471474, we evaluated the IC(50) of each inhibitor with a single experiment. The present work could be applied to the systematic study of biochemical binding and inhibition processes particularly in the field of mechanistic enzymology and the pharmaceutical industry.

Cell-Based Dose Responses from Open-Well Microchambers

Cell-based assays play a critical role in discovery of new drugs and facilitating research in cancer, immunology, and stem cells. Conventionally, they are performed in Petri dishes, tubes, or well plates, using milliliters of reagents and thousands of cells to obtain one data point. Here, we are introducing a new platform to realize cell-based assay capable of increased throughput and greater sensitivity with a limited number of cells. We integrated an array of open-well microchambers into a gradient generation system. Consequently, cell-based dose responses were examined with a single device. We measured IC50 values of three cytotoxic chemicals, Triton X-100, H2O2, and cadmium chloride, as model compounds. The present system is highly suitable for the discovery of new drugs and studying the effect of chemicals on cell viability or mortality with limited samples and cells.

Evaluation of peristaltic micromixers for highly integrated microfluidic systems

Microfluidic devices based on the multilayer soft lithography allow accurate manipulation of liquids, handling reagents at the sub-nanoliter level, and performing multiple reactions in parallel processors by adapting micromixers. Here, we have experimentally evaluated and compared several designs of micromixers and operating conditions to find design guidelines for the micromixers. We tested circular, triangular, and rectangular mixing loops and measured mixing performance according to the position and the width of the valves that drive nanoliters of fluids in the micrometer scale mixing loop. We found that the rectangular mixer is best for the applications of highly integrated microfluidic platforms in terms of the mixing performance and the space utilization. This study provides an improved understanding of the flow behaviors inside micromixers and design guidelines for micromixers that are critical to build higher order fluidic systems for the complicated parallel bio/chemical processes on a chip.

Wide Range Logarithmic Gradient Formation for Cell Response

Recently, the number of potential drug targets has dramatically increased because of the recent completion of the human genome sequencing and the progress in genomics and proteomics. In parallel, the number of new drugs for those targets has also been increased due to the use of combinatorial synthesis and the increased access to natural molecules [1]. However, this has not increased consequently the number of approved new drugs delivered to patients [2]. Indeed the drug discovery process is still limited by numbers of challenges; among them the need to analyze in more rapid and accurate manner precious sample of drug candidates.Copyright

Exponential Concentration Gradients in Microfluidic Devices for Cell Studies

Microscale technologies are a powerful tool in many biological and chemical applications, as they utilize only small reagent volumes. Microfluidics is especially well compatible with biological materials and applications, for example protein crystallization, high throughput assay analysis, and various cell studies. In that context, non-linear gradients of particles and molecules as well as efficient mixing of the components inside the lab-on-a-chip are crucial for many experimental studies: testing of and analyzing biological responses to different analyte concentration levels, studying the native cell microenvironment or cellular responses during different growth and proliferation stages. Thus, a microfluidic approach that allows for generation of different concentration gradients and specifically exponential gradients emerges as a helpful technology, and is also compatible with cells.© 2011 ASME