Transcript The Development of Bioluminescent Biosensors for Air Environment

Bioluminescent Sensors for Space Ecosystems
Li
1
Yang ,
2
Kratasyuk
Valentina
1SLSTP
Trainee, Carnegie Mellon University, Pittsburgh, PA, 15213,
2Professor of Biophysics, Krasnoyarsk State University, Krasnoyarsk, Russia
Objectives
Light Intensity Signal
These two bioluminescent tests were
assessed on the PerkinElmer Victor2 Bioluminometer along with
environmental samples from Kennedy Space Center (KSC).
Development of Bioluminescent
Test Systems
This experiment establishes standard experimental
procedures for conducting bioluminescent tests to
monitor contaminants in the water and air of closed
environments.
Effect of Common Toxins on Bioluminescent
Detection Systems
Methods
Results and Discussion
The standard reaction mixture of coupled enzyme system
was determined: 10 µl 0.002% tetradecanal, 5 µl 0.04 mM
FMN, 2 µl Luciferase-Oxidoreductase, 20 µl Phosphate
buffer, 10 µl NADH..
Bioluminescent Assessments of
Environmental Samples
Fig 1. Effect of Ethanol on E.coli Luminous Bacteria
Water Filtration Assessment
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Luminous Bacteria [10 µl]
Luminescencee
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Fig 6. Bioluminescent Assay of the Capacity of Filters to Eliminate
Bacteria
Luminous Bacteria [20 µl]
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Luminous Bacteria [80 µl]
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Luminous Bacteria [120 µl]
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Luminous Bacteria [140 µl]
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Fig. 3 Liquid Butenol on the Coupled Enzyme System
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Luminescence
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Buthenol Gas
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Acknowledgements
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The sensitivity of luminous bacteria and the enzyme
system to ethanol (1 µl) was more than for butanol (2
µl) (Fig. 2,3). Therefore, 1µl of ethanol would be
used for further experiments.
The bioluminescence tests found that 1/3 hoaglands nutrient
Obtaining the Coupled Enzyme Reaction Mixture water collected from the environmental chambers at KSC had
Fig 4. Influence of Ethanol on Aldehyde
no toxicity effects on the biological test system (Fig. 7);
filtered and unfiltered water showed the same light intensity
emissions.
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Soil Seed Medium Bioluminescent Assessment
A Nutrient Plant Soil Seed Medium was investigated by
a luminous bacteria assay (Fig. 13, 14).
Fig 5. Influence of Ethanol on FMN
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Fig 9. Luminescence of Concentrations of E.coli
Luminous Bacteria
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Fig 8. Optical Density of Concentrations of E.coli
Luminous Bacteria
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Light emission intensity was dependent on tetradecanal
aldehyde and FMN concentrations (Fig. 4, 5). Ethanol
was found to compete with tetradecanal as it reduced the
intensity of light emissions with increasing
concentrations of aldehyde. This suggests that ethanol
acts to disrupt tetradecanal activity in the enzymatic
reaction. Ethanol did not influence the FMN substrate
activity.
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Optical Density
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Fig. 14 Bioluminescent Assay of the Nutrient Seed Medium
(logarithmic)
Fig 13. Bioluminescent Assay of the Nutrient Seed Medium
(linear)
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Coupled Enzymatic Reaction:
NADH:FMN-oxidoreductase
NADH (NADPH) + H+ + FMN  NAD(NADP)+ + FMNH2 (1)
Luciferase
FMNH2 + RCHO + O2  FMN + RCOOH + H2О + h (2)
’
The biological systems consisted of bioluminescent bacteria and
their enzymatic extracts. Both test systems are based on the coupled
enzymatic reaction shown above.
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Luminescence
This research developed two bioluminescent test systems for toxicity
assays: the whole cell bacteria and the coupled enzyme system.
1. Farre M., Barcelo D. Toxicity Testing of waste water and sewage
sludge by Biosensors, bioassays and chemical analysis. Trends in
Analytical Chemistry, Vol. 22, No. 5, 2003.
2. Kratasyuk V.A. Esimbekova E.N. Polymeric Biomaterials, The
PBM Series, V.1:Introduction to Polymeric Biomaterials, Arshady R
Ed, Citus Books, London, pp. 301-343, 2003.
3. Kratasyuk V.A., et al. The use of bioluminescent bio-tests for study
of Natural and laboratory aquatic ecosystems. Chemosphere, 42: 909915, 2001.
4. Kratasyuk V. A., et al. Bioluminescent water quality monitoring of
salt lake Shira. Luminescence; 14: 193-195, 1999.
5. Paddle, Brian. Biosensors for Chemical and Biological agents of
defense Interest. Review Article. Biosensors and Bioelectronics Vol.
11 No. 11:1079-113, 1996.
6. Vetrova E., Bioluminescence characteristics of Lake Shira water.
Aquatic Ecology 36: 309-315, 2002.
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References
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Fig. 7 Bioluminescent Assessement of Growth Chamber Water Filtration
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Fig 12. Bioluminescent Assay of Butenol Gas on
Coupled Enzyme System
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Butanol gas was found to produce a slight inhibition of
the enzymatic reaction (Fig. 12).
Nutrient Water Bioluminescent Assessment
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Bioluminescent assessment of NanoCeram filters showed
that the filters were capable of filtering bacteria (Fig. 6).
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Filtered Bacteria
Bacteria Concentration
The bacterial system exposed to ethanol showed very
pronounced reduction in light intensity (Fig.1).
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Fig. 2 Liquid Buthenol on E.coli Luminous Bacteria
Fig 11. Bioluminescent Assay of Buthenol Gas on
E.coli Bacteria System
[Direct Injection ]
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• Bioluminescent methods were developed in this research to
monitor the air, water, and soil samples in closed ecosystems.
These methods serve as a set of guidelines for conducting
bioluminescent toxicity tests for closed environments.
• Bioluminescent tests were found to be capable of detecting
toxins in the liquid solutions.
• The bioluminescent systems were found to have high sensitivity
to minute amounts of liquid ethanol (1µl) and butanol (2ul),
which are common cleaners on space shuttles.
• New bioluminescent methods for control of the purification of
water were developed with NanoCeram filters.
• The coupled enzyme system was slightly inhibited by an air
sample of 500 ppm of buthanol gas.
These investigations of bioluminescent assays indicate the
advantages of using bioluminescence in applications for space
biotechnology. This result will be used to develop a proposal
entitled
“Bioluminescent
Biosensors
for
Space
biotechnology”. The future prospects of this research is directed
to the development of bioluminescent systems to control levels
of contaminants in the air, water, and soil of closed ecological
life support systems. In addition, bioluminescent methods for
control of water filtration will be developed in collaboration with
the Argonide Corporation.
Bioluminescent Assessment of Gasses
Gas samples (5000 ppm Butanol) were injected to the
bioluminescent systems by two methods: (1) the gases
were bubbled to 2 ml luminous bacteria solution (Fig.
10); (2) the gases were directly injected into the
microplate (Fig. 11). Butanol was found to have a slightly
excitatory effect on the bacteria.
Fig. 10 Bioluminescent Assay of Buthenol Gas on
E.coli Bacteria System
[Injection into Vial ]
Conclusions
Future Work
Soil Seed Medium: Bioluminescent Test of Seed Medium for Soil
The seed medium fiber was cut into pieces, massed from 0.1 to 0.7 g, and soaked in 10 ml of dionized water
overnight. 50 µl of E.coli bacteria was pipeted in concentrations from 1 to 10-5 into a microplate and the
initial light intensity was measured. 20 µl seed medium solution was injected into the bacteria after 1-2 min
and the new light intensity was recorded.
PerkinElmer Bioluminometer
This project aims to develop sensitive, low cost, versatile
bioluminescent sensors capable of monitoring multiple aspects of the
internal environment in closed ecological space life support systems.
The objectives for this study were six fold:
• to develop the biological component of bioluminescent sensors to
monitor closed environment of space ecosystems.
• to find the conditions (the amount of luminous bacteria and
concentrations of enzymes, flavin mononucleotide FMN,
tetradecanal aldehyde, NADH) to conduct environmental toxicity
assays
• to investigate the sensitivity of these bioluminescent test systems
on model pollutants (ethanol and buthanol)
• to investigate the nutrient water from environmental growth
chambers and the process of its purification with NanoCeram filters
• to develop the methods of gas pollutant detection
• to develop assays for control of water filtration systems in
environmental growth chambers
Gass: Bioluminescent Assessment of Toxic Gasses
Gas samples of 1000 ppm ethanol and 500 ppm buthanol were harnessed from the VOC project at the SLS
lab. E.coli with LUX-gene dissolved in tryptic soy broth solution was prepared in concentrations from 1 to
10-5. Gas samples were directly injected into the microplate and indirectly injected in a sealed 2 ml vial.
Light intensity I (t) was measured after definite time intervals for the duration of 1-2 min. The changes in
light intensity I(0)/I(t)*100 % were correlated with toxicity of the air samples to biological organisms.
Luminescence
As NASA embarks on a new era of human space exploration, the
environmental control of closed ecosystems will be
crucial for the long-term success of missions into
space since they maintain essential life support
functions to sustain a human crew during space
flight. It will be of crucial importance to develop
biological sensors to monitor the environmental
conditions inside closed ecosystems. The biological
monitoring
of
the
environmental
constituents of closed ecological systems
can be accomplished by bioluminescent
detection. In the past, bioluminescent
sensors have been developed to monitor
natural aquatic ecosystems [1,2,4,5,6].
These methods were adapted to monitor
systems used for human space travel.
Liquid: Bioluminescent Assessment of a Water Filtration System
The bioluminescent test system consisted of 100 µl E.coli bacteria in control in dilutions from 1 to 10-5of
the bacterial solution. 80 ml of 1/3 Hoaglands water was collected from KSC
growth chambers. Nutrient water was injected into the bioluminescent bacterial
solutions on the microplate. Light intensity readings were taken with the
bioluminometer to determine the steady state curve. A 5ml syringe with
NanoCeram filters from Argonide Co. was used to filter the growth chamber
water. Filtered water was pipeted on the microplate to see if bioluminescent test
system can assess the filtration of water. Bacterial solution was filtered with
NanoCeram and pipeted onto the microplate. The number of bacteria was calculated from their optical density at a 600 nm setting using a Genesis 20 spectrophotometer to be compared with
light intensity readings from the bioluminometer.
Bioluminescent Bacteria System
To find the volume for maximum light intensity, E.coli bacteria
dissolved in tryptic soy broth was pipeted in volumes from 10 to
140 µl into a microplate. The effect of different mediums [water
and ethanol] was tested with different volumes of liquids from 0 to
100 µl.
Coupled Enzyme Test System
The enzymatic reaction mixture contained 10 µl 0.002% Aldehyde
solution, 5 ul 0.04 mM FMN, 2-5 µl Luciferase-oxidoreductase (1
ml phosphate buffer added to vial of lyophilized enzymes), 20 µl
phostphate buffer pH 6.9, 10 µl NADH.
To determine effect of common toxins on the coupled ezyme
system, buthanol was injected to the reaction mixture in
concentrations from 0 to 10 µl.
To determine the reaction mixture, 1 µl ethanol was injected into
enzymatic reaction mixture with varying amounts of FMN and
Aldehyde.
Luminescence
Bioluminescent Sensors for Space Biotechnology
KSC Growth Chambers
Luminescence
Introduction
Bioluminescent Test Systems
Luminescence
Bioluminescent assays were conducted to monitor the toxicity of
contaminants in air, water, and soil samples taken from
environmental chambers located at the Space Life Science
Laboratory (SLSL) at Kennedy Space Center. Two methods were
developed to monitor contaminants in closed ecological systems.
They consisted of an in vivo assay using luminous bacteria, and an
in vitro assay using the coupled enzyme system NADH:FMNoxidoreductase-luciferase. The bioluminescent assays were used to
detect the contaminants in samples of water and gas. The luciferase
enzyme system was found to have more sensitivity to ethanol than
the bacteria system. Bioluminescent methods for the control of
liquid filters were developed with luminous bacteria.
Methods and Materials for Developing Bioluminescent Detection Systems
Luminescence
Abstract
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To determine if luminous bacteria could be used as an
accurate measure of water filtration, the luminescence of the
unfiltered bacteria (Fig. 9) was compared to their optical
density (Fig. 8). The comparison showed that luminous
bacteria test was highly sensitive.
The nutrient soil solution has no toxic effect on the
bioluminescent system. This proves to be good for using
the bioluminescent system for toxicity testing. These
results along with previous results also show that while
our biosensor is sensitive to toxins like ethanol and
butanol it is not affected by environmental materials like
nutrient water or soil seed mediums.
This research was conducted as a part of the 2005 Spaceflight and Life
Sciences Training Program funded by the National Aeronautics and
Space Administration. The authors recognize the support of the
Dynamac Corporation, the NASA Spaceflight and Life Sciences
Training Program Academic Partner Alliance and the United States
Department of Agriculture.
Thanks to Diane Shoeman, SIFT (Summer Industrial Fellowships for
Teachers) and Frank Mycroft (SLSTP trainee) for conducting parallel
laboratory research, Dr. Ignascio Eraso for providing samples of
buthanol and ethanol, Dr. Micheal Roberts and Michelle Birmele for
assistance with the PerkinElmer Victor 2 Bioluminometer, Lashelle E.
McCoy for providing samples of 1/3 hoaglands solution from
environmental growth chambers. In addition, we would like to thank
SLSTP trainees, Antrelle Kid, Jake Elmer, Jonathan for providing
laboratory materials essential for performing these experiments.