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
– Degradation of immune efficiency
– Atrophy of muscles
– 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
Date: 11-June-07
GeneSat System Architecture
Overall system concept
Tony Ricco, John Hines,
GeneSat team
Remarkable constraints…
• 2 kg total mass
PCB data
logger
• 2 W average power
• 2 L total volume
fluidic card
w/ heaters
PCB
LED/processing
… & requirements
• full autonomy
• 12 high-sensitivity blue-excited
fluorescence + optical density
units, < 30 cm3 (2 in3) each
• integrated fluidics and culture
wells, 10 expts. in 1.1 mL
• zero-power stasis of biology
• integral control measurements
• stable 1 atm, high RH, 34 ± 0.5
°C environment
media bag
optics
rails
pump,
valve
optics array
front support
GeneSat Payload System
Fluidic card
Date: 11-June-07
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
ALine
• Membrane filter at each well inlet and outlet
• Loaded pre-launch with E. coli in stasis medium
• 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
6.5 mm
channel
0.5 !m
membrane
Optically clear acrylic
2
Date: 11-June-07
GeneSat Payload System
Fluidic system
George Swaiss, David Oswell, Chris
Storment, Matthew Piccini
4 psi
28 kPa
Nutrient
evaporation
Fluidic card
Valve
0.5 psi
3.5 kPa
GeneSat Payload System
Optical system
Well w/ E. coli
(110 !L)
Saline/ waste
Date: 11-June-07
Linda Timucin, Tony Ricco, Stephane
Follonier, Peter Mrdjen, Bob Ricks,
Optical Research Associates, Optics One
Optical density
(light scattering)
green LED
3.5 mm
dia.
ellipsoid
Fluidic card
48 mm
Excitation
filter
Emission
filter
Intensity-tofrequency
detector
(TAOS TSL 237:
105 linear range)
48 X 34 X 18 mm =
29 cm3 (1.8 in3)
Fluorescent
excitation LED
(Luxeon: 1 W)
3
GeneSat Payload System
Sensors
Pressure Sensor
Temperature Sensor
Date: 11-June-07
Chris Storment, Bob
Ricks, Matthew Piccini
Relative Humidity
Sensor
Radiation 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:
• Optical absorbance (turbidity, OD)
• Viability indicator: Alamar Blue
• Colorimetric assay: metabolic products
cause blue dye " pink dye
S. cerevisiae
LAUNCH: 19 May 2009
Fluidic/Thermal/Optical Architecture
heater layer
PC board
LED
thermal spreader w/ T sensors
capping layer
capping layer
Gas-perm. membrane
Optical quality / clear
outlet
Acrylic
filter: 1 !m
4 mm
channel
yeast
7.7 mm
channel
Acrylic
filter: 1 !m
inlet
Gas-perm. membrane
Optical quality / clear
capping layer
capping layer
spreader w/ sensors
Detector chip
PC board
heater layer
3-color LED for
OD & viability:
track population
during growth,
viability using
Alamar Blue
indicator dye
Fluidic/
optical/
thermal
crosssection
Detector for OD
and viability
measurement
using 3-color
absorbance
6
PharmaSat Technology Architecture - 2
Fluidic, optical, & thermal layers
Solar panels
optical
PCB/excitn.
heater layer
thermal sprdr
Electronics
BFOT* card stack
fluidic card
fluid
storage
& delivery
Bus
Pressure vessel
thermal sprdr
heater layer
optical
PCB/detn.
*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
[ABoxidized]
(blue form)
[ABreduced]
(pink form)
Optical Density
(no. of cells)
8
PharmaSat Data (red
(red channel)
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. Mancinelli2, A. Mattioda, W. Nicholson3, R. Quinn2, 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
Washington University
2 SETI Institute
3 University of Florida/Kennedy Space Center
1 George
O/OREOS Dual-Payload Technology Architecture
Each P/L experiment-plus-instrument contained in a single 10-cm cube
opening
motor
electronics
UV-vis
spectrometer
Organics P/L
solar panels
LED PC board
biobiobioBlock Block Block
1
2
3
Bus
Detector board
electronics
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
•
Grow @ 35 – 37 °C for 1 – 17 days
•
Measure RGB transmittance @ 615, 525, 470 nm
Bacillus subtilis
– 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
»
»
»
»
Halorubrum
temperature (6 sensors per 12-well bioblock)
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
layer
radFET
space
radn.
PC board – 0.8 mm thick
LED
thermal spreader w/ T sensors
capping layer
PTFE membrane
capping layer
air
nucleopore
membrane
(hydrophobic)
2.8 mm
Polycarbonate
or ultem (polyamide)
nucleopore
membrane
(hydrophilic)
12 mm
Polycarb. + PVP
Gas-perm. membrane
Optical quality / clear
capping layer
capping layer
sapphire
Detector
spreader w/ sensors
radFET
heater layer
PC board
11
SESLO Integrated Fluidic System: 3 independent bioBlocks
bioBlock1
9 mm
pump
Growth
medium
A
V
V
Growth
medium
B
pump
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
O/OREOS Prototype & Flight Unit
SEVO
SESLO
Launch:
Fall, 2010
(Kodiak,
Alaska)
Conclusions
The tools of bio- and micro-technology combined with
automation & integration enable a range of biology
experiments in small space platforms
• Small satellites enable more experiments: space access, low cost
• Fundamental biological phenomena in a unique environment
• Human health & safety
• Origin, evolution, distribution, & future of life in the universe (astrobiology)
• Unique zero-shear-rate suspensions of cells & microorganisms
• Relevance to terrestrial medicine & pharma development
• Accelerated test platform: osteoporosis, muscle atrophy, immune
impairment, radiation effects
• Novel conditions impact microorganism function including metabolic
processes, secreted proteins
• Spaceflight environment increases the virulence of some pathogens
• Growth of ultra-low-defect-density protein crystals
13