Nimisha Srivastava

Nanoliter viscometer for analyzing blood plasma and other liquid samples

We have developed a microfabricated nanoliter capillary viscometer that quickly, easily, and inexpensively measures the viscosity of liquids. The measurement of viscosity is based on capillary pressure-driven flow inside microfluidic channels (depth approximately 30 microm and width approximately 300 microm). Accurate and precise viscosity measurements can be made in less than 100 s while using only 600 nL of liquid sample. The silicon-glass hybrid device (18 mm by 15 mm) contains on-chip components that measure the driving capillary pressure difference and the relevant geometrical parameters; these components make the nanoliter viscometer completely self-calibrating, robust, and easy to use. Several different microfabricated viscometers were tested using solutions with viscosities ranging from 1 to 5 cP, a range relevant to biological fluids (urine, blood, blood plasma, etc.). Blood plasma samples collected from patients with the symptoms of hyperviscosity syndrome were tested on the nanoliter capillary viscometer to an accuracy of 3%. Such self-calibrating nanoliter viscometers may have widespread applications in chemical, biological, and medical laboratories as well as in personal health care.

Fully Integrated Microfluidic Platform Enabling Automated Phosphoprofiling of Macrophage Response

The ability to monitor cell signaling events is crucial to the understanding of immune defense against invading pathogens. Conventional analytical techniques such as flow cytometry, microscopy, and Western blot are powerful tools for signaling studies. Nevertheless, each approach is currently stand-alone and limited by multiple time-consuming and labor-intensive steps. In addition, these techniques do not provide correlated signaling information on total intracellular protein abundance and subcellular protein localization. We report on a novel phosphoFlow Chip (pFC) that relies on monolithic microfluidic technology to rapidly conduct signaling studies. The pFC platform integrates cell stimulation and preparation, microscopy, and subsequent flow cytometry. pFC allows host-pathogen phosphoprofiling in 30 min with an order of magnitude reduction in the consumption of reagents. For pFC validation, we monitor the mitogen-activated protein kinases ERK1/2 and p38 in response to Escherichia coli lipopolysaccharide (LPS) stimulation of murine macrophage cells (RAW 264.7). pFC permits ERK1/2 phosphorylation monitoring starting at 5 s after LPS stimulation, with phosphorylation observed at 5 min. In addition, ERK1/2 phosphorylation is correlated with subsequent recruitment into the nucleus, as observed from fluorescence microscopy performed on cells upstream of flow cytometric analysis. The fully integrated cell handling has the added advantage of reduced cell aggregation and cell loss, with no detectable cell activation. The pFC approach is a step toward unified, automated infrastructure for high-throughput systems biology.

Studies of Phosphorylation During Innate Immune Signaling using On-Chip Cell Preparation and Downstream Flow Cytometry

Fine temporal resolution is required for monitoring protein phosphorylation events key to cellular signaling pathways. With the goal of teasing apart the kinetics of phosphorylation cascades central to the human innate immune response to pathogen invasion, our group has developed a microfluidic device that integrates flow cytometry with required upstream cell preparation steps. Streamlined cell preparation and analysis allows monitoring of events with kinetic resolution on the order of minutes - not tens of minutes to hours. The planar microfluidic device contains 50 mm long spiral mixers, porous polymer monoliths for selective exchange of reagents, and incubation chambers (~1000 cells per chamber) where macrophage cells (RAW264.7) are challenged with a chemical signal of Gram-negative bacteria (lipopolysaccharide) and subsequently labeled with fluorescent immunoreagents. Finally, on the same device, the labeled macrophage cells are analyzed using two-color flow cytometry. Such an integrated self-contained microfluidic platform promises to be of widespread use to host- pathogen studies in infectious disease laboratories.