Transcript The Development of Bioluminescent Biosensors for Air

The Development of Bioluminescent Biosensors for Air
Environment Monitoring in Closed Ecosystems
Li Yang, 2005 SLSTP Trainee
Carnegie Mellon University
June 23, 2005
Dr. Valentina Kratasyuk, Principal Investigator
Biophysical Department, Krasnoyarsk State University (Russia)
Introduction
Future Space Travel
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NASA missions rely on closed
ecological life support systems
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Monitoring toxicity in closed
ecological system is a problem
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Physical/chemical tests can only
tell us chemical composition
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Biological assays of the toxicity of
environment must be developed
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We suggest Bioluminescence
Glow to Grow Experiments
Bioluminescent Biosensors for Space Biotechnology
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Bioluminescence - biological emission of light in
enzymatic reactions with luciferase
Bioluminescence property of living organisms gives rapid
response rate to toxicity
Bioassays will be used to measure biological toxicity
Hypothesis
•The prediction of this project
is that bioluminescent systems
will respond to the toxicity of
environmental conditions in
closed ecological systems.
•Contaminants in the
environment that are toxic to
live organisms will act as
inhibitors and interrupt
bioluminescent reactionscausing measurable reduction
in light intensity
Bioluminescent Biosensors
Convert biological light emissions to electrical signals
Plant stem
Plant stem
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Bioluminescent sensors: toxicity assay for living organisms,
highly sensitive and accurate bioassay, small, portable,
simple, and low cost, rapid response rate, quantitative
Physical/chemical tests (gas-chromatography and massspectroscopy etc.) : will not reveal whether substance is
harmful to living organism and require large complicated
devices
Materials and Methods
1. Initial Air Sampling from Mars Green
House and Environmental Chambers
2. Bioluminescent Bioassay in vivo
Luminous Bacteria Assay:
Photobacterium phosphoreum
3. Bioluminescent Bioassay in vitro
Coupled Enzyme System: NADH-
FMN:Oxidoreductase-luciferase
4. Test NanoCeram Filters with
Bioluminescent Biosensors in H20 and Air
Materials and Methods
Experimental Reagents
Materials
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E.coli recombinant
LUX-gene from
luminous bacteria
E.coli recombinant
(LUX-gene)
Tryptic Soy Broth
NaCl – 3% solution
Luciferase
Oxidoreductase
Tetradecanal
NADH
FMN
Ethanol
Phosphate buffer pH 7.0
Enzymes
NADH-FMNoxidoreductaseluciferase
Bioluminometer
Luminous Bacteria
Membrane
Inhibitors
Ag chlorine
Heavy metals,
Polar agents,
Phenols
solvents,
alcohols
Wall
Cytoplasmic
membrane
Heavy metals
(cadmium),
Phenolics
(Dichlorophenol)
R
FMN + NADH + H+
FMNH2 + R1COH + O2
+
Protonmotive
force
Non-polar organics
(Toluene)
Oxidative phosphorylation
uncouplers
(phentachlorophenol, SDS)
L
FMNH2 + NAD+
FMN + R1COOH + H2O
h
490 nm
Cytoplasmic
constituents
Membrane
ATPase
Electron transport system Respiratory blockers
eg cyanide
Aldehydes,
Cationic agents,
Heavy metals
Luminous Bacteria Assay
Scheme of Analysis
Luminous
bacteria,
Enzymes
control
luminescence Iс
test
luminescence Iе
sample
Luciferase Enzymatic Reactions
Coupled Enzyme System: oxidoreductase-luciferase
Bioluminescence is the emission of light, produced from a
chemical reaction, which originates within a living organism
H+
NAD(P)*
FMNH2
Luciferase
Oxidoreductase
NAD(P)H
O2
RCHO
FMN
RCOOH
H2O
NADH:FMN-oxidoreductase
NADH (NADPH) + H+ + FMN 
NAD(NADP)+ + FMNH2 (1)
luciferase
FMNH2 + RCHO + O2  FMN + RCOOH + H2О + h (2)
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Analysis of Luminescent Intensity
Bacterial Indexes (BI) calculated from the Luminous Bacteria Assay
Luciferase Indexes (LI) calculated from the Coupled Enzyme System
Sampling
I,mV
Maximum Emission (I0)
I0 I
s
time, s
Sampling Emission (Is)
LI
=
BI
=
I s / I0
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NanoCeram Filters
Bioluminescent sensors will test whether the NanoCeram Filters
remove the contaminants in liquid solutions.
We will take a liquid solutions samples.
Attach the filter and push the liquid solutions through the filter
Bioluminescence Biosensors will be used to test the samples before
and after filtrations.
Expected Outcomes
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To clarify whether
bioluminescent biotests should
be recommended as the alarm
test to control acute toxicity from
a variety of sources such as air,
water or soil samples in Closed
Ecological Systems.
To design bioluminescent
biosensors for control of the air
and water quality surrounding
plants grown in closed
environments (the toxicity of the
gas and liquid phases ) using
the bioluminescent organisms or
their enzymatic reactions
To live and work on Mars with
my Biosensors
Pitfalls and Limitations
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Challenging new methods
Unknown effects of gases on bioluminescence
Using the plates instead of cuvettes in bioluminometer.
Air samples must be loaded into micro-tray. We are not
sure if the air samples can be directly injected into the
tray.
Timetable of Scheduled Activities
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Week 1: Introduction to Experiment and General
Research
June 13-19
Week 2: Experimental Design and Operation of
Bioluminometer
June 20-26
Week 3 & Week 4 : Continue Glowing Experiments
June 27-10
Week 5: Data analysis, Poster preparations, and ASGSB
abstracts,
July 11-17
Week 6: Finale Poster Presentations, Dinner Banquet
SLSTP
July 18-24
Acknowledgements
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Dr. Valentina Kratasyuk, Prof. Biophysical Department, Krasnoyarsk State
University (Russia)
Dr. Sergey Gusev, Biophysical Department,
Krasnoyarsk State University (Russia)
Diane Shoeman
SIFT Employee from Merritt Island High School
Dr. Ray Allen Bucklin, Prof., Agriculture and Biological Engineering
Department, University of Florida
Dr. Melanie Correll, Asst Prof., Agriculture and Biological Engineering
Department, University of Florida
Program Directors
SLSTP (Spaceflight and Life Sciences Training Program)
Project Counselors
SLSTP (Spaceflight and Life Sciences Training Program)
Elizabeth Raffi
SLSTP (Spaceflight and Life Sciences Training Program)
All SLSTP Trainees
SLSTP (Spaceflight and Life Sciences Training Program)
References
1.
Kratasyuk V.A. etc. The use of bioluminescent biotests for study
of natural and laboratory aquatic ecosystems. Chemosphere, 42 (2001)
909-915.
2.
Kratasyuk V.A. Esimbekova E.N. Polymeric Biomaterials, The
PBM Series, V.1:Introduction to Polymeric Biomaterials, Arshady R
(Ed), Citus Books, London 2003, pp 301-343
3.
Paddle, Brian. Biosensors for Chemical and Biological agents of
defense interest. Review Article. Biosensors and Bioelectronics Vol. 11
No. 11 pp. 1079-113, 1996
4.
Farre M., Barcelo D. Toxicity Testing of water and sewage sludge
by Biosensors, bioassays and chemical analysis. Trends in Analytical
Chemistry, Vol. 22, No. 5, 2003.
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