BIOLUMINESCENT SENSORS

1. BIOLUMINESCENT SENSORS JING WANG Department of Nutrition and Food Science ENPM808B Dec 3rd, 2003

2. Outline • Structure of Biosensor • Bioluminescent bacteria • Target Analytes • Transducers • Applications • Summary

3. Structure of Biosensor

4. Analyte Transducer Bio- Receptor Measurable Signal

5. Bioluminescent bacteria

6. firefly luciferase luciferin + ATP + O2 oxyluciferin + PPi + CO2 + h Mg2+ (max = 560nm) Firefly Bioluminescence

7. Photobacterium phosphoreum Xenorhabdusnematophilus Bacterial Bioluminescence • Vibrio • Photobacterium • Xenorhabdus

8. Bioluminescence luxCDABE Genes

9. synthetase RCOOH + ATP + NADPH NADP + AMP + Ppi + RCHO reductase transferase RCOX + HOH(HSR’) RCOOH(RCOSR’) + XH luciferase FMNH2 + RCHO + O2 FMN +H2O + RCOOH +h(490nm) Figure 1. Bacterial bioluminescence pathway (adapted from Van Dyk, 1998)

10. Figure 2. cloning of bioluminescent gene into E. coli strains

11. Target Analytes

12. Mercury Hg Potassium nitrate KNO3 Nickel Ni Inorganic Substances

13. Phenol Benzene Urea Octane Ethanol Naphthalene Organic Substances

14. Transducer

15. Photomultipier Tubes

16. Turner BioSystems' TD-20/20 single-tube luminometer Luminometer

17. Applications

18. Quantitating loss of bioluminescence due to the toxicity of the sample tested or of the environmental condition imposed Light out The choice of the promoter driving expression of the lux genes determines the specificity of the response Light on

19. 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

20. CCPAHs PCPAHs Phenanthrene Pyrene Naphthalene Anthracene Benzo[a]pyrene 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.

21. Immobilization Procedure Recombinant E. Coli Strain RFM443 Materials and methods

22. Ampicillin 100g/ml 20 ml Agar Media 500 l fresh LB medium Collected Cells E. Coli GC2 cells 10 mm Polypropylene tubes Materials and methods Centrifuge 6000rpm 10 min 25 ºC 50 ml sample Sterile glass beads (0.05 g, 150 to 212 m) 100 l cell mixture

23. Immobilization Procedure Recombinant E. Coli Strain RFM443 Measurement System Materials and methods Solubilization of PAHs Using Rhamnolipids as Biosurfactant

24. Materials and methods Schematic diagram of the soil biosensor system

25. Results and Discussion Relative Bioluminescence (RBL) The ratio of the test bioluminescence to the control’s bioluminescence

26. Results and Discussion Bioluminescent response to PCPAHs (a) pyrene (b) benzo[a]pyrene

27. Results and Discussion Bioluminescent response to CCPAHs (a) naphthalene (b) anthracene

28. Results and Discussion Bioluminescent response to CCPAHs (c) phenanthrene

29. 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.

30. 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

31. Green Fluorescence Protein (GFP) Xenorhabdus luminescens (Photorhabdus luminescens) Background

32. Divergent Orientation Tandem Orientation Figure 3. Fusion gene constructs used in this study Materials and Methods two stress-responsive Escherichia coli biosensor strains

33. Hydrogen Peroxide Cadmium Chloride Materials and Methods Hydroxyl radical-forming chemicals

34. Methyl-N-nitro-N-nitrosoguanidine (MNNG) Mitomycin C Materials and Methods Genotoxins

35. Isopropanol Ethanol Phenol CH3CH2OH Materials and Methods General toxincants

36. 100 l 100 l chemical 100 l chemical opaque Plate luminometer E. coli strains clear 100 l 96-well plate FLx800 Microplate fluorometer 250 ml flask 50 ml LB medium

37. 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

38. 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

39. 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)

40. Conclusions • Both strains showed an induction of green fluorescent protein (GFP) and bioluminescence when they experienced DNA and oxidative damage, respectively.

41. 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.

42. 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.

43. Summary

44. Advantages • Quick response time • Not sensitive to environmental changes • Easy to operate and control

45. Disadvantages • Difficult to remain the cell alive and viable • Not very stable during the sensing time • Less specific comparing to other types of biosensors

46. THANK YOU!