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

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

3. Structure of Biosensor

4. Transducer Bio- Receptor Analyte Measurable Signal

5. Bioluminescent bacteria

6. Firefly Bioluminescence firefly luciferase luciferin + ATP + O 2 oxyluciferin + PPi + CO 2 + h Mg 2+ ( max = 560 nm)

7. Bacterial Bioluminescence Vibrio Photobacterium Xenorhabdus Photobacterium phosphoreum Xenorhabdus nematophilus

8. Bioluminescence luxCDABE Genes

9. transferase RCOX + HOH(HSR’) RCOOH(RCOSR’) + XH synthetase RCOOH + ATP + NADPH NADP + AMP + Ppi + RCHO reductase FMNH 2 + RCHO + O 2 FMN +H 2 O + RCOOH +h (490nm) luciferase 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. Inorganic Substances Mercury Hg Potassium nitrate KNO 3 Nickel Ni

13. Organic Substances PhenolBenzene Urea Octane EthanolNaphthalene

14. Transducer

15. Photomultipier Tubes

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

17. Applications

18. 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

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. 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 CCPAHs Pyrene Benzo[a]pyrene PCPAHs

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

22. Materials and methods Ampicillin 100  g/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

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

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

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

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

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

35. Materials and Methods IsopropanolPhenolEthanol CH 3 CH 2 OH General toxincants

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

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 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

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