Transcript BIOLUMINESCENT SENSORS - A. James Clark School of

BIOLUMINESCENT
SENSORS
JING WANG
Department of Nutrition and Food Science
ENPM808B Dec 3rd, 2003
Outline
Structure of Biosensor
Bioluminescent bacteria
Target Analytes
Transducers
Applications
Summary
Structure of
Biosensor
Analyte
BioReceptor
Transducer
Measurable
Signal
Bioluminescent
bacteria
Firefly Bioluminescence
firefly luciferase
oxyluciferin + PPi + CO2 + h
luciferin + ATP + O2
Mg2+
(max = 560 nm)
Bacterial Bioluminescence
Vibrio
Photobacterium
Photobacterium
phosphoreum
Xenorhabdus
Xenorhabdus
nematophilus
Bioluminescence luxCDABE Genes
synthetase
RCOOH + ATP + NADPH
NADP + AMP + Ppi + RCHO
reductase
luciferase
transferase
RCOX + HOH(HSR’)
RCOOH(RCOSR’) + XH
FMNH2 + RCHO + O2
FMN +H2O + RCOOH +h(490nm)
Figure 1. Bacterial bioluminescence pathway
(adapted from Van Dyk, 1998)
Figure 2. cloning of bioluminescent gene into E. coli strains
Target Analytes
Inorganic Substances
Mercury Hg
Potassium
nitrate KNO3
Nickel Ni
Organic Substances
Phenol
Octane
Benzene
Ethanol
Urea
Naphthalene
Transducer
Photomultipier
Tubes
Luminometer
Turner BioSystems' TD-20/20 single-tube luminometer
Applications
Light out
Quantitating loss of bioluminescence
due to the toxicity of the sample
tested or of the environmental
condition imposed
Light on
The choice of the promoter driving
expression of the lux genes determines
the specificity of the response
Example 1
Monitoring and classification of PAH
toxicity using an immobilized
bioluminescent bacteria
Hyun Joo Lee, Julien Villaume, David C. Cullen,
Byoung Chan Kim, Man Bock Gu. Biosensors and
Bioelectronics, Volume 18, Issues 5-6, May 2003,
Pages 571-577
Background
Polycyclic Aromatic Hydrocarbons (PAHs)
PAHs are a class of very stable organic molecules
made up of only carbon and hydrogen. These
molecules are flat, with each carbon having three
neighboring atoms much like graphite.
CCPAHs
Naphthalene
Phenanthrene Anthracene
PCPAHs
Pyrene
Benzo[a]pyrene
Materials and methods
Recombinant
E. Coli Strain
RFM443
Immobilization
Procedure
Materials and methods
Ampicillin
100g/ml
Centrifuge
6000rpm
50 ml sample
E. Coli GC2 cells
Polypropylene
tubes
10 min
500 l fresh
25 ºC
LB medium
Sterile glass beads
(0.05 g, 150 to 212 m)
20 ml Agar
Media
Collected
Cells
10 mm
100 l cell
mixture
Materials and methods
Recombinant
E. Coli Strain
RFM443
Solubilization of
PAHs Using
Rhamnolipids as
Biosurfactant
Immobilization
Procedure
Measurement
System
Materials and methods
Schematic diagram of the soil biosensor system
Results and Discussion
Relative Bioluminescence (RBL)
The ratio of the test
bioluminescence to the control’s
bioluminescence
Results and Discussion
Bioluminescent
response to
PCPAHs
(a) pyrene
(b) benzo[a]pyrene
Results and Discussion
Bioluminescent
response to
CCPAHs
(a) naphthalene
(b) anthracene
Results and Discussion
Bioluminescent response to CCPAHs (c) phenanthrene
Conclusions
The response patterns of this soil
biosensor system to CCPAHs or
PCPAHs were clearly identifiable.
Only CCPAHs were found to cause
toxicity and inhibit cellular metabolism,
while PCPAHs did not affect any
changes in bioluminescence responses.
Example 2
Construction and characterization of
novel dual stress-responsive
bacterial biosensors
Robert J. Mitchell and Man Bock Gu.
Biosensors and Bioelectronics, In Press,
Corrected Proof, Available online 18
November 2003
Background
Green Fluorescence Protein
(GFP)
Xenorhabdus luminescens
(Photorhabdus luminescens)
Materials and Methods
two stress-responsive Escherichia coli
biosensor strains
Divergent Orientation
Tandem Orientation
Figure 3. Fusion gene constructs used in this study
Materials and Methods
Hydroxyl radical-forming chemicals
Hydrogen Peroxide
Cadmium Chloride
Materials and Methods
Genotoxins
Mitomycin C
Methyl-N-nitro-Nnitrosoguanidine
(MNNG)
Materials and Methods
General toxincants
Isopropanol
Ethanol
CH3CH2OH
Phenol
100 l
100 l chemical
opaque
Plate luminometer
250 ml flask
50 ml LB medium
100 l chemical
E. coli strains
clear
100 l
96-well plate
FLx800 Microplate
fluorometer
Results and Discussion
Figure 4. Time-dependent plots of the fluorescent response from DUO-1
after exposure to various concentrations of (a) mitomycin C and (b) MNNG
Table1. Response characteristics of DUO-1 and DUO-2
a Concentration (mg/l) giving the maximum induction
b NR: no response; RBL or FL value of less than 2.0 and 1.25, respectively
c Value in parenthesis is the lowest concentration (mg/l) giving a twofold
induction of bioluminescence or a maximum slope of 0.01
Figure 5. Time-dependent bioluminescent plots from DUO-1 (a and c) and
DUO-2 (b and d) after exposure to various concentrations of hydrogen
peroxide (a and b) and mitomycin C (c and d)
Conclusions
Both strains showed an induction of
green fluorescent protein (GFP) and
bioluminescence when they
experienced DNA and oxidative
damage, respectively.
Conclusions
The tandem orientation of the two fusion
genes within DUO-2 allowed it to sensitively
respond to genotoxins via the production of
bioluminescence. The characteristics of
DUO-2's bioluminescent response to each
stress were easily distinguishable, making it
useful for the detection of both stresses.
Conclusions
Furthermore, tests with mixtures of
chemicals showed that both DUO-1
and DUO-2 were responsive when
chemicals causing oxidative or
genotoxic stress were present as a
single chemical or within complex
chemical mixtures.
Summary
Advantages
Quick response time
Not sensitive to environmental
changes
Easy to operate and control
Disadvantages
Difficult to remain the cell alive
and viable
Not very stable during the sensing
time
Less specific comparing to other
types of biosensors