Biosensors, Bugs, and Biodefense

Transcription

1 Biosensors, Bugs, and Biodefense Daniel V. Lim, Ph.D. Distinguished University Professor Division of Cell Biology, Microbiology, and Molecular Biology Department of Biology and Center for Biological Defense University of South Florida

2 September 11, 2001

3 Anthrax and ricin contamination of letters

4 Homeland Security Presidential Directives 7 & 9 Protection of Critical Infrastructure

5 CDC Bioterrorism Agent List Category A: Bacillus anthracis (anthrax) ersinia pestis (plague) Smallpox virus Ebola virus Category B: E. coli O157:H7 Ricin Category C: Multi-drug resistant Mycobacterium tuberculosis

6 Limitations of Current Technology to Detect Bioterrorism Agents Time-consuming Requires pure or clean samples Sophisticated equipment Not suitable for field use Requires trained scientific personnel Not very accurate

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8 Translational Research Laboratory Development Application Government Industry Universities Microbiology Molecular Biology Immunology Biochemistry

9 Evanescent Wave Biosensors (Naval Research Laboratory) RAPTOR (Research Int l) Array Biosensor (Hanson Technologies) Integrating Waveguide (Creatv MicroTech)

10 How Biosensors Detect Pathogens Step 1: Attach Capture Antibody to Fiber Optic Waveguide Step 2: Apply Sample. Target Organism Binds to Waveguide Step 3: Wash Away Other Cells and Debris Step 4: Add Specific Detection Antibody Step 5: Turn on Laser. Detection Antibody Fluoresces, Indicating a Positive Sample.

11 Evanescent Wave Technology Cy5 Excited Cy5 >650 nm signal Evanescent wave }100 nm }100 nm Waveguide

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13 Fiber Optic Waveguide Array Waveguide

14 Array Biosensor 6 x 6 Array Image Ricin Cholera Tularemia Brucellosis Anthrax SEB

15 Biosensor Assays Analytes Detected Bacillus anthracis Vaccinia (smallpox) SEB toxin Ricin Vibrio cholerae Complex Matrices Processed Ground beef Alfalfa sprouts Lettuce wash Apple juice Recreational water E. coli O157:H7 Enterococcus Mycobacterium tuberculosis Salmonella typhimurium Cryptosporidium parvum Potable water Human saliva Human blood Human feces Powder

16 Recovery of Live Bacteria and PCR Analysis after Biosensor Assay PCR and genetic analysis Remove waveguide from assay chamber Inoculate selective enrichment broth and incubate Isolate pure culture/ antibiotic resistance

17 Polymerase Chain Reaction (PCR) Products from Fiber Optic Waveguides 10 4 cells 10 5 cells 10 6 cells 10 7 cells

18 Green Fluorescent Protein E. coli O157:H7 for Assay Optimization pglo plasmid mediated GFP detection GFP-labeled E. coli detected on plates and waveguides

19 GFP-Labeled E. coli O157:H7 on Waveguides Fiber Optic Waveguide Array Waveguide

20 Capture Efficiency of E. coli O157:H7 on Waveguides 10 5 cells/ml 10 6 cells/ml 10 7 cells/ml RAPTOR % Capture % Surface Area Array Sensor % Capture % Surface Area

21 Fluorescent In Situ Hybridization (FISH) DNA oligonucleotide probe HO-3 C C T T T C G A A T G 5 -PO 4 PO 4-5 U C G G A A A G C U U A C U G A ribosomal RNA (rrna) target 3 -OH

22 FISH Probes Label Cells Stringency controlled by: temperature, formamide, salt concentration

23 Waveguide Current Method Antibodies used to capture bacteria Reporter Abs must be used FISH Method Reporter Probes already in cells Evanescent wave detection distance ~ 100 nm External C5 outside of range Internal C5 within range

24 FISHing in the Advanced Biosensors Laboratory

25 Affinity-Based Molecules used in Biosensor Assays Antibodies Gangliosides Aptamers Lectins Artificial antibodies (ScFvs( ScFvs) Phage display Peptides Antimicrobials Phage display ScFv Antimicrobials

26 Biosensor Assay Development Biosensor assay Detection of Gram negative bacteria (E. coli and Salmonella) Antibody alternative LPS binding peptide (Polymyxin B) Detection of Gram Bacteriophage-specific Positive bacteria binding protein (Staphylococcus (Protein 17) aureus and Bacillus anthracis)

27 Molecules That Bind LPS Lipopolysaccharide (LPS) O antigen Lipid A Polymyxin B

28 E. coli Capture by Polymyxin B or Antibody in Biosensor Assays E. coli O157:H7 E. coli Gram negative Capture using Polymyxin B E. coli O157:H7 S. aureus Gram positive Capture using antibody

29 Bacteriophage-Specific Binding Proteins S. aureus -HRP Protein 17-His 6 Substrate Fluorescent product 525 S. aureus (Host cell) E. coli O157:H7 622 Protein 17 Crude lysate

30 Rapid Biosensor Monitoring of Potable and Recreational Water Automated, continuous monitoring of water for biothreat agents and microbial pathogens

31 Bacteria In Filter Backflush Pump Pump Carboy Pump Backflush reservoir Orbital Shaker Forward Pump Eluted Concentrate (Bacteria out) to drain

32 Automated Concentration System (ACS) (Patent Pending) NaOH Reservoir Filter Water Input Concentrate

33 Rapid Detection of Enterococci: Ben T. Davis Beach

34 Dead End Filtration Dead End Clamp Filter Pump Beach water Pressure Gauge Permeate

35 Filter Backflush Clamp Filter Pump Clamp Clamp Elution Buffer Pressure Gauge Concentrated Sample Out (containing target)

36 Recreational Dead End Concentrator (Rec DEC) Filtration set-up in hatch of car Tubing exiting car to collect water sample

37 Rec DEC (cont d) Battery Pack Peristaltic pump Hollowfiber Battery Filter Pack Permeate Collection

38 Indirect Immunodetection Cells directly adsorbed to a polystyrene waveguide. Unlabeled 1 o IgG Ab added. Laser on Cy-5 labeled anti-igg 2 o Ab binds to 1 o Ab and fluoresces when laser is activated.

39 Determination of Water Quality with the Rec DEC-RAPTOR Procedure DOH Water Quality Category Poor Moderate Good Concentration of Enterococci (CFU/100 ml) > < 35 Change in Signal (pa( pa) from RAPTOR assay ~ N/A (no detection)

40 Detection Times: Standard Method vs. Rec DEC-RAPTOR Procedure Method Time to Detection (h) EPA Method 1600: Rec DEC-RAPTOR Procedure: Collection, elution and preparation Centrifugation Indirect biosensor assay and data analysis Total:

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42 Rapid Biosensor Detection of E. coli O157:H7 in Spinach

43 Biosensor Applications Bioterrorism/biowarfare agents (anthrax, plague, smallpox, toxins) (U.S. Army, NIH) Airborne pathogens (post office, sick building syndrome) (USPS) Waterborne pathogens (Cryptosporidium,( E. coli, Salmonella, cholera) (PCU, EPA) Foodborne pathogens (E.( coli O157:H7, Salmonella, Listeria) ) (FDA, USDA) Infectious diseases (blood, feces) Recovery of live bacteria for viability confirmation, DNA analysis, and forensic evidence

44 Agencies Supporting Biosensor Research: U.S. Army/RDECOM National Institutes of Health (SBIR) National Science Foundation (SBIR, STTR) U.S. Environmental Protection Agency Water Environment Research Foundation Pinellas County Utilities Southern California Water Coastal Water Research Project Cooperative Institute for Coastal and Estaurine Environmental Technology (CICEET) National Cattlemen s s Beef Association

45 Corporate, Government, and University Partners EPA

46 Joyce Marianne Betty Sonia Stephaney Sonja Sarah Kim Alicia