Integrated Microfluidic Systems in Challenging Environments: - PDF document

Integrated Microfluidic Systems in Challenging Environments: Biological Studies in Earth Orbit Tony Ricco NASA Ames Research Center, Moffett Field, CA On leave from Stanford University O/OREOS GeneSat PharmaSat Life Science Studies in Space: Why, How? • One of NASA’s missions is human exploration of the solar system – Study space effects at fundamental biological level to develop strategies/therapies – In-situ experiments: immediate, accurate info. ( vs . sample return) – Microgravity plus complex space radiation environment can’t be simulated on Earth – Modern life science research is compatible with small, autonomous payloads – Small satellites offer frequent, inexpensive space access as 2° payloads • Deleterious effects of space travel are relevant to health on Earth – Loss of bone density – Atrophy of muscles – Degradation of immune efficiency – Radiation damage – Some biological effects are accelerated in space: unique insights into their mechanisms could lead to new or more effective therapies • First Mission: GeneSat-1 demonstrated real-time measurement of gene expression levels in autonomous 5-kg satellite in Earth orbit GeneSat-1 model organism: E. coli • ~1 x 2 ! m bacteria • survive nutrient deprivation in dormant state over wide temp. range (4 – 37 °C) until stable orbit • GFP fusions track expression of key genes • fluorescent assay of GFP levels • optical density measurement for population estimate 1

GeneSat System Architecture Date: 11-June-07 Overall system concept Tony Ricco, John Hines, GeneSat team Remarkable constraints… • 2 kg total mass PCB data logger • 2 W average power fluidic card w/ heaters • 2 L total volume PCB … & requirements LED/processing media bag • full autonomy optics pump, rails • 12 high-sensitivity blue-excited valve fluorescence + optical density units, < 30 cm 3 (2 in 3 ) each • integrated fluidics and culture wells, 10 expts. in 1.1 mL • zero-power stasis of biology optics array • integral control measurements • stable 1 atm, high RH, 34 ± 0.5 °C environment front support GeneSat Payload System Date: 11-June-07 Fluidic card David Oswell, Tony Ricco, Chris Storment, Matthew Piccini, Leanna Levine • 12-well culture-and-analysis plate • 10 assay wells, 2 control/standard wells • 110 ! L/well ! 1.1 mL total on-card volume • Reservoir capacity ~15 mL • Membrane filter at each well inlet and outlet • Loaded pre-launch with E. coli in stasis medium ALine • Infused upon stable ( g , T) orbit with glucose solution to initiate growth Gas-permeable membrane (applied after culture inoculation) 3.3 mm Bio-zone channel channel 6.5 mm 0.5 ! m membrane Optically clear acrylic 2

GeneSat Payload System Date: 11-June-07 Fluidic system George Swaiss, David Oswell, Chris Storment, Matthew Piccini 4 psi evaporation 28 kPa Nutrient Valve Fluidic card 0.5 psi Saline/ waste 3.5 kPa GeneSat Payload System Date: 11-June-07 Optical system Linda Timucin, Tony Ricco, Stephane Follonier, Peter Mrdjen, Bob Ricks, Optical Research Associates, Optics One Optical density (light scattering) Well w/ E. coli green LED (110 ! L) 3.5 mm dia. Fluidic card ellipsoid 48 mm Excitation Emission filter filter Intensity-to- frequency 48 X 34 X 18 mm = detector 29 cm 3 (1.8 in 3 ) (TAOS TSL 237: 10 5 linear range) Fluorescent excitation LED (Luxeon: 1 W) 3

GeneSat Payload System Date: 11-June-07 Sensors Chris Storment, Bob Ricks, Matthew Piccini Pressure Sensor Temperature Sensor Relative Humidity Radiation Sensor Sensor (PIN diode) Analog Devices 590 Motorola MPXH6101A Hamamatsu S3071 3-Axis Accelerometer Silicon Designs 1221-002 4

GeneSat Launch: 16 Dec 2006 420 km, 90 min orbits; re-entry/disintegration 04-August-2010 GeneSat-1: Comparing flight with ground control 5

PharmaSat: Effect of Microgravity on Yeast Susceptibility to Antifungal Drugs Tony Ricco, Macarena Parra, John Hines, Mike McGinnis, Dave Niesel, Matthew Piccini, Linda Timucin, C. Friedericks, E. Agasid, C. Beasley, M. Henschke, C. Kitts, A. Kudlicki, E. Luzzi, D. Ly, I. Mas, M. McIntyre, R. Rasay, R. Ricks, K. Ronzano, D. Squires, J. Tucker, B. Yost NASA Ames, UTMB, Santa Clara U. • Grow yeast cells in multiwell fluidics card in microgravity • Measure efficacy of antifungal agent to inhibit growth of fungus • Control + 3 concentrations of antifungal • 12 wells each for statistics • Measure cell health & growth: S. cerevisiae • Optical absorbance (turbidity, OD) • Viability indicator: Alamar Blue • Colorimetric assay: metabolic products cause blue dye " pink dye LAUNCH: 19 May 2009 Fluidic/Thermal/Optical Architecture heater layer 3-color LED for PC board OD & viability: LED track population thermal spreader w/ T sensors during growth, capping layer capping layer viability using Gas-perm. membrane Optical quality / clear Alamar Blue outlet indicator dye filter: 1 ! m 4 mm Fluidic/ Acrylic channel optical/ yeast 7.7 mm thermal channel Acrylic filter: 1 ! m cross- inlet section Gas-perm. membrane Optical quality / clear capping layer capping layer Detector for OD spreader w/ sensors and viability measurement Detector chip using 3-color PC board absorbance heater layer 6

PharmaSat Technology Architecture - 2 Fluidic, optical, & thermal layers Solar panels optical PCB/excitn. heater layer thermal sprdr Electronics fluidic card Bus thermal sprdr fluid heater layer storage optical BFOT* card stack & delivery PCB/detn. Pressure vessel *BFOT = Biology/Fluidics/Optical/Thermal PharmaSat Fluidics Card • Micronics’ approach to PharmaSat fluidics card – laser-cut acrylic layers – roller laminated using pressure-sensitive adhesive same adhesive proven on GeneSat 7

PharmaSat Fluidic Mixing, Dilution, & Delivery System Alamar Blue concentrations & OD calculated from RGB absorbances including spectral overlap corrections [ [ AB AB oxidized oxidized ] ] (blue form) (blue form) [ AB AB reduced ] [ reduced ] (pink form) (pink form) Optical Density (no. of cells) 8

PharmaSat Data ( red channel PharmaSat Data ( red channel ) ) • Antifungal effect is clear in both Spaceflight and Ground • Response for High Antifungal consistent with metabolism being less suppressed in ! gravity than on Earth… but cell division remains suppressed 9

Organism/ORganics Exposure to Orbital Stresses: The O/OREOS NanoSatellite A. J. Ricco, D. Squires, J. W. Hines, P. Ehrenfreund, 1 R. Mancinelli 2 , A. Mattioda, W. Nicholson 3 , R. Quinn 2 , O. Santos Science support: N. Bramall, J. Chittenden, K. Bryson, A. Cook, M. Parra, D. Ly Development by the NASA-Ames Nanosatellite Engineering Team NASA Ames Research Center 1 George Washington University 2 SETI Institute 3 University of Florida/Kennedy Space Center O/OREOS Dual-Payload Technology Architecture Each P/L experiment-plus-instrument contained in a single 10-cm cube solar panels opening LED PC board motor bio- bio- bio- Block Block Block Bus 1 2 3 electronics UV-vis Detector board spectrometer electronics Organics P/L Biology P/L Bus 10

Payload 1: Space Environment Survivability of Live Organisms (SESLO) Astrobiology: Origin, evolution, distribution, & future of life in the universe • Two organisms, wildtype & mutant, exposed to ! gravity & space radiation – < 10 -3 g – 2 – 20 Gy total dose (6 months in 650 km orbit) • Dry organisms on ! well walls pre integration • Rehydrate & feed 6 ! wells / organism: t = 1 wk , 3 mo , 6 mo Bacillus subtilis • Grow @ 35 – 37 °C for 1 – 17 days • Measure RGB transmittance @ 615 , 525 , 470 nm – track culture population via optical density (both organisms) – track metabolic activity via Alamar Blue ( B. subtilis only) • Sensors: T , p , RH , rad (integrated dose), ! grav » temperature (6 sensors per 12-well bioblock) Halorubrum chaoviatoris » pressure, relative humidity (1 sensor each) » radiation total dose @ both ends of wells (2 radFETs) » microgravity levels calc’d. from solar panel currents SESLO (bio) Fluidic/Thermal/Optical Architecture Fluidic / optical / thermal cross-section heater space layer radn. PC board – 0.8 mm thick radFET radFET LED thermal spreader w/ T sensors PTFE membrane capping layer capping layer air nucleopore membrane 2.8 mm (hydrophobic) Polycarbonate 12 mm or ultem (polyamide) nucleopore membrane (hydrophilic) Polycarb. + PVP Gas-perm. membrane Optical quality / clear capping layer capping layer sapphire spreader w/ sensors heater layer Detector radFET radFET PC board 11

SESLO Integrated Fluidic System: 3 independent bioBlocks bioBlock1 9 mm Growth Growth medium medium pump V V pump A B 75 ! L per well NC solenoid valves open 2x/day to maintain fluid back-pressure to compensate for evaporation from wells throughout organism growth period !"!#$%&'#%()*+,-%!.//012 34+--)= 1),+7-%7; >1)**.1) ?)**)5@ !"#$"%&' ($'#&( 34)1/05 67-8175 10=C"3* 9.*%:-8)1;0<) A0=+0B7-%!4+)5= 12