Microfluidics

STMC scientists Anup Singh and Conrad James are developing and validating Sandia’s Microfluidics or lab-on-a-chip technology that allows biologists within the center to study cell signaling at the level of single cells—a feat not achievable by traditional biochemical methods. The platform, known as Microscale Cell and Immune Analysis (MICA) platform (Figure 1), enables understanding both the actions of single cells and the interactions of these cells with their environments. MICA seamlessly integrates cell culture and handling, cell stimulation (e.g., introduction of a pathogenic challenge), fluorescence-activated cell sorting (FACS), flow cytometry, high-resolution imaging, and antibody-based proteomic analysis. All experimental manipulations are carried out at the microscale and are fully automated, providing exquisitely precise control over each cell and its environment. Because microscale experiments consume vanishingly small amounts of cells and reagents, MICA can be used to investigate cellular processes that have proven impossible or impractical to study at conventional scale such as work with primary cells and with clinical samples.

MICA offers exceptional features and enables measurements that are not possible with conventional assays, as shown in the following table:

MICA is being used for two specific applications in STMC—1) for imaging single cells using the iCellator module and 2) for profiling of protein phosphorylation and inflammatory mediator (histamine, cytokines) secretions in single cells in ProteoFlow chip as described below.

1. iCellator Chip (Investigator: Conrad James): This chip, easily interfaced with an epifluoresence or TIRF microscope, enables long-term (days to weeks) imaging of live cells trapped at microfabricated features. The chip contains multiple imaging chambers for running control and challenge experiments simultaneously within a single chip. Positioning cells at pre-defined locations enables cells to be optimally interfaced to optical detectors and to be subjected to controlled environmental conditions for real-time monitoring of cell signaling events (Figure 2). The chips have up to 100 parallel cell trap structures for holding cells in place, and the cell trap configuration also places cells immediately adjacent to the coverslip for high-resolution optical interrogation. A key feature of the traps is that cells within each trap are fluidically-isolated in a local environment. This allows each cell to be isolate from secreted substances (e.g. cytokines) released by other cells.

2. ProteoFlow Module (Investigator: Anup Singh ) We have developed a novel chip (Figure 3) that integrates automated cell manipulation, correlated imaging, and subsequent flow cytometric analysis for temporally- and spatially-resolved analyses (Figure 4) of phosphoproteins, cytokines, and other proteins in single cells. The chip can be programmed by a user to automate process steps for cell stimulations (including dosing, rapid mixing, and timed incubations); antibody preparatory steps (including cell fixation, plasma membrane permeabilization, fluorescent immuno-staining and numerous intermediate washing steps); and subsequent flow cytometry.

Publications:

1. Srivastava N, Brennan JS, Renzi RF, Wu MY, Branda SS, Singh AK, Herr AE. Fully Integrated Microfluidic Platform Enabling Automated Phosphoprofiling of Macrophage Response. Analytical Chemistry 81, 3261-3269 (2009).

2. James, Conrad D. and Moorman, Matthew W. and Carson, Bryan D. and Branda, Catherine S. and Lantz, Jeffrey W. and Manginell, Ronald P. and Martino, Anthony and Singh, Anup K. Nuclear translocation kinetics of NF-kappa B in macrophages challenged with pathogens in a microfluidic platform. Biomedical Microdevices, 11 (3). pp. 693-700 (2009).

3. James, CD, N Reuel, ES Lee, RV Davalos, SS Mani, A Carroll-Portillo, R Rebeil, A Martino, C Apblett. (2008). Impedimetric and optical interrogation of single cells in a microfluidic device for real-time viability and chemical response assessment. Biosensors and Bioelectronics 23:845–861.

4. Perroud, TD, JN Kaiser, JC Sy, TW Lane, CS Branda, AK Singh, KD Patel. (2008). Microfluidic-based cell sorting of Francisella tularensis infected macrophages using optical forces. Analytical Chemistry 80:6365–6372.

5. Perroud, TD; Meagher RJ; Kanouff, MP; Renzi, RF; Wu, M; Singh, AK; Patel, KD. Isotropically Etched Radial Micropore for Cell Concentration, Immobilization, and Picodroplet Generation. Lab on a Chip 9, 507-515 (2009). Featured on journal back cover.

For more information on Sandia’s microfluidics capabilities, please visit http://www.sandia.gov/microfluidics/

For more information on MICA platform, please visit http://www.sandia.gov/mica/