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Thomas D. Perroud, Ph.D.

Research in Microfluidic-based Instrumentation for Single-Cell Analysis

Thomas Perroud photo

Biographical Information

Dr. Thomas Perroud is a Senior Member of the Technical Staff at Sandia National Laboratories in the Department of Biosystems Research and Development. Dr. Perroud has been involved in numerous projects developing microfluidic-based bioanalytical instruments for cell sorting, flow cytometry, single-cell imaging, and cell preparation for studying host-pathogen interactions. He is also working on actinides extraction and purification, and single-molecule detection on chip. He has collaborated with researchers in the departments of Bioenergy and Defense Technology, Advanced Systems Engineering & Deployment, Energy Nanomaterials, and Combustion Chemistry, as well as with researchers at UTMB and UCSF.

In 1999, Dr. Perroud earned a bachelor’s degree in chemical engineering at the Ecole Polytechnique Fédérale de Lausanne (EPFL, Switzerland), followed by a Diploma in 2000 under the joint-direction of Prof. Horst Vogel at EPFL and Prof. Richard N. Zare at Stanford University. During his Diploma, Dr. Perroud developed a vesicle fusion assay to characterize synaptic transmission using single-molecule fluorescence spectroscopy, confocal spectroscopy, and laser tweezers. In 2006, Dr. Perroud received a Ph.D. in physical chemistry at Stanford University in the laboratory of Prof. Richard N. Zare, where he developed the photon counting histogram for one-photon excitation, a single-molecule fluorescence spectroscopy technique that can identify conformational states in proteins. During his Ph.D., he was awarded a Roche Research Foundation fellowship as well as a Franklin Veatch Memorial fellowship.

Dr. Perroud’s postdoctoral appointment at Sandia under the direction of Dr. Kamlesh Patel was part of the MicroScale Immune and Cell Analysis project - a multidisciplinary project aimed at characterizing the role of two bacterial pathogens, F. tularensis and Y. pestis, in the interruption or subversion of the human innate immune system’s response. As a member of the engineering team, he focused on the development of a microfluidic platform geared towards the full characterization of macrophage cells activated by the pathogen F. tularensis. More specifically, he co-developed a microfluidic-based fluorescence-activated cell sorter (µFACS) using optical forces as a deflection mechanism. Furthermore, he integrated this cell sorting functionality with a downstream single-cell array to combine two fundamentally different, but complementary, analytical techniques – high-throughput flow cytometry and high-resolution single-cell microscopy imaging. During his postdoctoral tenure, he received the Post-Doctoral Technical Showcase Outstanding Poster award.

Current and Former Research Projects at Sandia

A systems biology approach to viral hemorrhagic fever pathogenesis.

We are investigating whether pathological cytokine production in response to arenavirus infection is an infected cell-intrinsic (only that cell) or extrinsic (involving other cells) effect. Using novel fluorescent transcriptional reporters and in situ solid phase immunodetection, we are comparing cytokine production by cell populations and by isolated cells trapped in a microfluidic-based single-cell array.

Team members: OA Negrete, M Wu, CS Branda, BD Carson, and NK Herzog (UTMB).

Integration of radial micropores in microfluidic channels for cell concentration, immobilization, and picodroplet generation.

To enable several on-chip cell handling operations in a fused-silica substrate, we have radially embedded small shallow micropores in larger deeper microchannels using an adaptation of single-level isotropic wet etching. By varying the distance between features on the photolithographic mask, we can precisely control the overlap between two etch fronts and create a zero-thickness semi-elliptical micropore (e.g. 20 _m wide, 6 _m deep). To illustrate the wide range of cell handling operations enabled by micropores, we investigated three on-chip functionalities: continuous-flow particle concentration, immobilization of single cells, and picoliter droplet generation.

Movies: Micropore formation by overlapping etch fronts; On-chip particle concentration

Team members: RJ Meagher, MP Kanouff, RF Renzi, M Wu, AK Singh, and KD Patel.

Microfluidic-based cell sorting of macrophages using optical forces.

We have extended the principle of optical tweezers as a noninvasive technique to actively sort hydrodynamically focused cells based on their fluorescence signal in a microfluidic device. To demonstrate the selective nature of optical sorting, we isolated a subpopulation of macrophages highly infected with the fluorescently labeled pathogen Francisella tularensis subsp. novicida.

Movies: Optical sorting of macrophage cells; Principle of _µFACS

Team members: JN Kaiser, JC Sy, TW Lane, CS Branda, AK Singh, and KD Patel.

Optimization of signal-to-noise in a deployable pathogen detection device.

We are optimizing the signal-to-noise ratio for the detection of protein biotoxins (e.g. ricin, Shiga toxin, Staphylococcal Enterotoxin B, and Botulinum toxin) using laser-induced fluorescence and electrophoretic immunoassays in a microfluidic chip. Our goal is to produce a low-cost, portable, rapid diagnostic device suitable for the multiplexed detection of biotoxins in the case of a bioterror attack.

Team members: AV Hatch, JS Brennan, RF Renzi, and AK Singh.

Rapid raw sample identification of actinides using a portable detection platform.

We are designing and engineering a portable microfluidic-based platform for the extraction and detection of actinides in “real-world” samples.

Team members: K Rahimian, RF Renzi, MS Derzon (Sandia), and M Kaminski, C Mertz (Argonne National Laboratory).

On-chip immunohistochemistry for fluorescence microscopy followed by quantification by micro flow cytometry.

We combine microscopy imaging and flow cytometry on a single chip to examine temporal differences in innate immune signaling pathways upon pathogen exposure.

Team members: M Wu, N Srivastava (UCSB), CS Branda, RF Renzi, and AK Singh.

Microchannel electrophoresis with single-molecule detection.

We have coupled microfluidic protein separations with the unique time-resolved confocal fluorescence microscope developed by Dr. Carl Hayden at Sandia’s Combustion Research Facility. This microscope is able to perform single-molecule lifetime and spectral measurements, fluorescence correlation spectroscopy, and fluorescence cross-correlation spectroscopy. These capabilities, which have been used to study protein–protein and antibody–toxin interactions, are expected to be useful for a variety of sophisticated diagnostics.

Team members: RJ Meagher, CC Hayden, and AK Singh.

Selected Publications

Lee, S. E., Sasaki, D. Y., Perroud, T. D., Yoo, D., Patel, K. D., & Lee L. P. Biologically Functional Cationic Phospholipid-Gold Nanoplasmonic Carriers. JACS (in press).

Perroud, T. D.; Meagher R. J.; Kanouff, M. P.; Renzi, R. F.; Wu, M.; Singh, A. K.; Patel, K. D. Isotropically Etched Radial Micropore for Cell Concentration, Immobilization, and Picodroplet Generation. Lab on a Chip 9, 507-515 (2009). Featured on journal back cover.

Perroud, T. D.; Kaiser, J. N.; Sy, J. C.; Lane, T. W.; Branda, C. S.; Singh, A. K.; Patel, K. D. Microfluidic-Based Cell Sorting of Francisella tularensis Infected Macrophages using Optical Forces. Analytical Chemistry 80, 6365-6372 (2008). Highlighted in Biophotonics International Magazine.

Perroud, T. D., Bokoch, M. P. & Zare, R. N. Cytochrome c conformations resolved by the photon counting histogram: Watching the alkaline transition with single-molecule sensitivity. Proceedings of the National Academy of Sciences of the United States of America 102, 17570-17575 (2005). Highlighted in Analytical Chemistry.

Perroud, T. D., Huang, B. & Zare, R. N. Effect of bin time on the photon counting histogram for one-photon excitation. ChemPhysChem 6, 905-912 (2005).

Huang, B., Perroud, T. D. & Zare, R. N. Photon counting histogram: One-photon excitation. ChemPhysChem 5, 1523-1531 (2004).

Perroud, T. D., Huang, B., Wallace, M. I. & Zare, R. N. Photon counting histogram for one-photon excitation. ChemPhysChem 4, 1121-1123 (2003).

Patents

Perroud, T. D., R. J. Meagher, K. D. Patel; Controlled pore formation in microchannels using overlapping single-level wet-etch fronts. (filed Jan. 2008, 61/062,545).

Perroud, T. D., K. D. Patel; A method for enhanced and selective immobilization of single cells. (filed Jan. 2008, 61/062,401).

Selected Presentations, Conference Proceedings

Perroud, T.D., C.S. Branda, N.H. Romano, A.K. Singh and K.D. Patel, On-chip Flow Cytometry Coupled to Single-Cell Imaging for Studying Signal-Transduction Pathways, in 23rd International Symposium on MicroScale Bioseparations. 2009 Boston (MA).

Perroud, T.D., R.J. Meagher, M.P. Kanouff, R.F. Renzi, M. Wu, A.K. Singh and K.D. Patel, Isotropically Etched Radial Micropore for Cell Concentration, Immobilization, and Picodroplet Generation, in Post-Doctoral Technical Showcase. 2008 Livermore (CA).

Perroud, T.D., C.S. Branda and K.D. Patel. Micropore Formation using Overlapped Etch Fronts in Integrated Devices for Cellular Analysis. in The 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences. 2008 San Diego (CA).

Perroud, T.D., J.N. Kaiser, J.C. Sy, T.W. Lane, C.S. Branda, A.K. Singh and K.D. Patel, Microfluidic Cell Sorting Using Photonic Forces for Host-Pathogen Studies, in 59th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy. 2008 New Orleans (LA).

Perroud, T.D., J.C. Sy and K.D. Patel, Integrated Microfluidic Discovery Platform for Cellular Analysis of Host-Pathogen Interaction in Macrophage Cells, in 31st International Symposium on Capillary Chromatography & Electrophoresis. 2007 Albuquerque (NM).

Perroud, T.D. and K.D. Patel. Rapid Fluorescence-Activated Cell Sorting with Optical-Force Deflection in a Microfluidic Device. in 21st International Symposium on MicroScale Bioseparations. 2007 Vancouver (Canada).

Perroud, T.D. and K.D. Patel. Rapid Fluorescence-Activated Cell Sorting with Optical-Force Deflection in a Microfluidic Device. in 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences. 2006. Tokyo (Japan).

Perroud, T.D., M.P. Bokoch, B. Huang, S. Kim and R.N. Zare, The Photon Counting Histogram: Monitoring Cytochrome c Conformations, in 49th Annual Meeting of the Biophysical Society. 2005 Long Beach (CA).

Perroud, T.D., B. Huang, M.I. Wallace and R.N. Zare. Photon Counting Histogram for One Photon Excitation. in International Symposium on Elucidating Biomolecular Networks by Single-Molecule Technologies. 2003 Ascona (Switzerland).

Perroud, T.D., M.I. Wallace, B. Huang, C. Parnot, B.K. Kobilka and R.N. Zare, Observation of b2-adrenergic receptor activation using a photon counting histogram, in 47th Annual Meeting of the Biophysical Society. 2003 San Antonio (TX).

Contact Information

Dr. Thomas D. Perroud
Department of Biosystems R&D
Sandia National Laboratories
PO Box 969, MS 9292
Livermore, CA 94551-0969

thomas.perroud@sandia.gov
phone: (925) 294-3133
fax: (925) 294-3020