BIOLUMINESCENT SENSORS

JING WANG

Department of Nutrition and Food Science

ENPM808B Dec 3rd, 2003

Outline

Structure of BiosensorBioluminescent bacteriaTarget AnalytesTransducersApplicationsSummary

Structure of Biosensor

TransducerBio-Receptor

Analyte

MeasurableSignal

Bioluminescent bacteria

Firefly Bioluminescence

firefly luciferase

luciferin + ATP + O2 oxyluciferin + PPi + CO2 + h

Mg2+ (max = 560 nm)

Bacterial Bioluminescence

Vibrio

Photobacterium

Xenorhabdus

Photobacterium phosphoreum

Xenorhabdus nematophilus

Bioluminescence luxCDABE Genes

transferase

RCOX + HOH(HSR’)

RCOOH(RCOSR’) + XH

synthetase

RCOOH + ATP + NADPH NADP + AMP + Ppi + RCHO

reductase

FMNH2 + RCHO + O2

FMN +H2O + RCOOH +h(490nm)

luciferase

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 SubstancesPhenol Benzene Urea

Octane Ethanol 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.

Naphthalene Phenanthrene Anthracene

CCPAHsPyrene Benzo[a]pyrene

PCPAHs

Materials and methods

Recombinant E. Coli Strain

RFM443

ImmobilizationProcedure

Materials and methodsAmpicillin 100g/ml

E. Coli GC2 cells

50 ml sample

Centrifuge

6000rpm

10 min

25 ºC Collected

Cells

500 l fresh

LB medium

20 ml Agar

Media

100 l cell mixture

10 mm

Sterile glass beads

(0.05 g, 150 to 212 m)

Polypropylene

tubes

Materials and methods

Recombinant E. Coli Strain

RFM443

ImmobilizationProcedure

Solubilization ofPAHs Using

Rhamnolipids asBiosurfactant

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 Methodstwo stress-responsive Escherichia coli biosensor strains

Figure 3. Fusion gene constructs used in this study

Divergent Orientation

Tandem Orientation

Materials and Methods

Hydrogen Peroxide Cadmium Chloride

Hydroxyl radical-forming chemicals

Materials and Methods

Mitomycin C Methyl-N-nitro-N-nitrosoguanidine

(MNNG)

Genotoxins

Materials and Methods

Isopropanol PhenolEthanol

CH3CH2OH

General toxincants

250 ml flask50 ml LB medium

E. coli strains

Plate luminometer

FLx800 Microplate fluorometer

100 l100 l chemical

opaque

100 l

100 l chemical

96-well plate

clear

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 inductionb NR: no response; RBL or FL value of less than 2.0 and 1.25, respectivelyc 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 timeNot sensitive to environmental

changesEasy 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