Biosensors, Bugs, and BiodefenseBiosensors, Bugs, and Biodefense Daniel V. Lim, Ph.D. Distinguished...
Transcript of Biosensors, Bugs, and BiodefenseBiosensors, Bugs, and Biodefense Daniel V. Lim, Ph.D. Distinguished...
Biosensors, Bugs, and Biosensors, Bugs, and BiodefenseBiodefense
Daniel V. Lim, Ph.D.Distinguished University Professor
Division of Cell Biology, Microbiology, and Molecular BiologyDepartment of Biology andCenter for Biological DefenseUniversity of South Florida
September 11, 2001September 11, 2001
Anthrax and Anthrax and ricinricin contamination contamination of lettersof letters
Homeland Security Presidential Directives 7 & 9Homeland Security Presidential Directives 7 & 9Protection of Critical InfrastructureProtection of Critical Infrastructure
CDC Bioterrorism Agent ListCDC Bioterrorism Agent ListCategory A:Category A:
Bacillus Bacillus anthracisanthracis (anthrax)(anthrax)YersiniaYersinia pestispestis (plague)(plague)Smallpox virusSmallpox virusEbola virusEbola virus
Category B:Category B:E. coli E. coli O157:H7O157:H7RicinRicin
Category C:Category C:MultiMulti--drug resistant drug resistant Mycobacterium tuberculosisMycobacterium tuberculosis
Limitations of Current Technology to Limitations of Current Technology to Detect Bioterrorism AgentsDetect Bioterrorism Agents
TimeTime--consumingconsumingRequires pure or clean samplesRequires pure or clean samplesSophisticated equipment Sophisticated equipment Not suitable for field useNot suitable for field useRequires trained scientific Requires trained scientific personnelpersonnelNot very accurateNot very accurate
MicrobiologyMicrobiologyMolecular BiologyMolecular BiologyImmunologyImmunologyBiochemistryBiochemistry
Government
UniversitiesIndustry
Translational ResearchLaboratory → Development → Application
Evanescent Wave BiosensorsEvanescent Wave Biosensors(Naval Research Laboratory)(Naval Research Laboratory)
RAPTOR (Research Int’l)
Array Biosensor (Hanson Technologies)
Integrating Waveguide (Creatv MicroTech)
Step 1: Attach Capture Antibody to Fiber Optic WaveguideStep 2: Apply Sample. Target Organism Binds to WaveguideStep 3: Wash Away Other Cells and DebrisStep 4: Add Specific Detection AntibodyStep 5: Turn on Laser. Detection Antibody Fluoresces, Indicating a Positive Sample.
Y Y Y Y YY YY Y YYYY YYYY
Y Y Y YY YY YY
Y Y YY Y YY Y
Y Y
How Biosensors Detect Pathogens
>650 nm
signal
ExcitedCy5
Cy5
Evanescent Wave Technology
Waveguide
Evanescent wave }100 nm
}100 nm
Array Waveguide
Fiber Optic Waveguide
Array Biosensor
6 x 6 Array Image
RicinRicinCholeraCholeraTularemiaTularemiaBrucellosisBrucellosisAnthrax Anthrax SEBSEB
Biosensor AssaysBiosensor Assays
Bacillus Bacillus anthracisanthracisVacciniaVaccinia (smallpox)(smallpox)SEB toxinSEB toxinRicinRicinVibrioVibrio choleraecholerae
E. coli E. coli O157:H7O157:H7EnterococcusEnterococcusMycobacterium Mycobacterium tuberculosis tuberculosis Salmonella Salmonella typhimuriumtyphimuriumCryptosporidium Cryptosporidium parvumparvum
Ground beefGround beefAlfalfa sproutsAlfalfa sproutsLettuce washLettuce washApple juiceApple juiceRecreational waterRecreational water
Potable waterPotable waterHuman salivaHuman salivaHuman bloodHuman bloodHuman fecesHuman fecesPowderPowder
Analytes Detected
Complex Matrices Processed
Recovery of Live Bacteria and PCR Recovery of Live Bacteria and PCR Analysis after Biosensor AssayAnalysis after Biosensor Assay
Remove waveguide from assay chamber
Inoculate selective enrichment broth
and incubate
PCR andgenetic analysis
Isolate pure culture/antibiotic resistance
Polymerase Chain Reaction (PCR) Products Polymerase Chain Reaction (PCR) Products from Fiber Optic Waveguidesfrom Fiber Optic Waveguides
104 cells
105 cells
106 cells
107 cells
Green Fluorescent ProteinGreen Fluorescent Proteinfor Assay Optimizationfor Assay Optimization
E. coliE. coli O157:H7O157:H7pGLOpGLO plasmid mediated plasmid mediated GFPGFP detectiondetectionGFPGFP--labeled labeled E. coli E. coli detected on plates and detected on plates and waveguideswaveguides
GFPGFP--Labeled Labeled E. coli E. coli O157:H7 O157:H7 on Waveguideson Waveguides
Fiber Optic Waveguide
Array Waveguide
Capture Efficiency of Capture Efficiency of E. coli E. coli O157:H7 on WaveguidesO157:H7 on Waveguides
4.974.970.420.420.040.04% Surface Area% Surface Area
0.450.450.350.350.410.41% Capture% Capture
Array SensorArray Sensor
0.400.400.070.070.010.01% Surface Area % Surface Area
0.310.310.540.541.231.23% Capture% Capture
RAPTORRAPTOR10107 7 cells/mlcells/ml10106 6 cells/mlcells/ml10105 5 cells/mlcells/ml
Fluorescent In Situ Hybridization (FISH)
PO4-5’3’-OH
U C G G A A A G C U U A C U G A
5’-PO4HO-3’C C T T T C G A A T G
DNA oligonucleotide“probe”
ribosomal RNA (rRNA)“target”
FISH Probes Label Cells
Stringency controlled by:temperature,formamide,salt concentration
Waveguide
FISH Method
Reporter Probes already in cells
Current Method Antibodies used tocapture bacteria
Reporter Abs must be used
Evanescent wave detection distance ~ 100 nm
External CY5 outside of range
Internal CY5 within range
FISHing in the Advanced Biosensors Laboratory…
AffinityAffinity--Based Molecules used in Based Molecules used in Biosensor AssaysBiosensor Assays
AntibodiesAntibodiesGangliosidesGangliosidesAptamersAptamersLectinsLectinsArtificial antibodies (Artificial antibodies (ScFvsScFvs))Phage displayPhage displayPeptidesPeptidesAntimicrobialsAntimicrobials
Phage display
ScFv
Antimicrobials
Biosensor Assay DevelopmentBiosensor Assay DevelopmentBiosensor assayBiosensor assay Antibody alternativeAntibody alternative
Detection of Gram Detection of Gram LPS binding peptideLPS binding peptidenegative bacteria negative bacteria ((PolymyxinPolymyxin B)B)((E. coli andE. coli andSalmonellaSalmonella))
Detection of Gram Detection of Gram BacteriophageBacteriophage--specificspecificPositive bacteria Positive bacteria binding proteinbinding protein((StaphylococcusStaphylococcus (Protein 17)(Protein 17)aureusaureus and and Bacillus Bacillus anthracisanthracis) )
Molecules That Bind LPSMolecules That Bind LPS
Polymyxin B
Lipopolysaccharide(LPS)
O antigen
Lipid A
E. coli E. coli Capture by Capture by PolymyxinPolymyxin B or B or Antibody in Biosensor AssaysAntibody in Biosensor Assays
E. coliO157:H7
Y
YCapture using Polymyxin B
E. coliO157:H7
YCapture using antibody
E. coliGram negative
S. aureusGram positive
BacteriophageBacteriophage--Specific Binding ProteinsSpecific Binding Proteins
S. aureus(Host cell)
S. aureus
Protein 17-His6
-HRPSubstrate
Fluorescentproduct
990
677
525
622
849
651
613
645
Protein 17Crude lysate
E. coliO157:H7
990
677
525
622
849
651
613
645
Protein 17Crude lysate
E. coliO157:H7
Rapid Biosensor Monitoring of Rapid Biosensor Monitoring of Potable and Recreational WaterPotable and Recreational Water
Automated, continuous monitoring of water for biothreat agents and microbial pathogens
Pump
Forward Pump
Pump
Backflush PumpFilter
Backflush reservoir
Bacteria In
Orbital Shaker
Carboy
Eluted Concentrate (Bacteria out)
to drain
Automated Concentration System (ACS)Automated Concentration System (ACS)(Patent Pending)(Patent Pending)
Filter
Water Input
NaOHReservoir
Concentrate
Rapid Detection of Rapid Detection of EnterococciEnterococci: : Ben T. Davis BeachBen T. Davis Beach
Dead End FiltrationDead End Filtration
Pump
Pressure Gauge
FilterClamp
Permeate
Beach water
Dead End
Filter BackflushFilter Backflush
Pump
Pressure Gauge
Filter
Clamp
Concentrated Sample Out (containing
target)
Elution Buffer
Clamp Clamp
RecRecreational reational DDead ead EEnd nd CConcentrator (Rec DEC)oncentrator (Rec DEC)
Tubing exiting car to collect water sample
Filtration set-up in hatch of car
Rec DEC (contRec DEC (cont’’d)d)
Battery Pack
Peristaltic pump
Battery Pack
Hollow-fiber Filter
Permeate Collection
Indirect Indirect ImmunodetectionImmunodetection
YYY YY YY YYY YY YY
YYY YY YY YYY YY YY
YYY YY YY
Y Y YY YYY
Laser
on
Cy-5 labeled anti-IgG 2o Ab binds to 1o Ab and fluoresces when laser is activated.
Cells directly adsorbed to a polystyrene waveguide.
Unlabeled 1o IgG Ab added.
Determination of Water Quality with Determination of Water Quality with the Rec DECthe Rec DEC--RAPTOR ProcedureRAPTOR Procedure
<< 3535
3636--104104
>> 105105
Concentration of Concentration of Enterococci Enterococci
(CFU/100 ml)(CFU/100 ml)
N/A (no detection)N/A (no detection)GoodGood
300300--500500ModerateModerate
~1000+~1000+PoorPoor
Change in Signal (Change in Signal (pApA) ) from RAPTOR assayfrom RAPTOR assay
DOH Water DOH Water Quality Quality
CategoryCategory
Detection Times: Detection Times: Standard Method Standard Method vs.vs.
Rec DECRec DEC--RAPTOR ProcedureRAPTOR Procedure
3.003.00Total:Total:
1.751.75Indirect biosensor assay and data Indirect biosensor assay and data analysisanalysis
0.500.50Centrifugation Centrifugation
0.750.75Collection, elution and preparationCollection, elution and preparation
Rec DECRec DEC--RAPTOR Procedure:RAPTOR Procedure:
24.0024.00EPA Method 1600:EPA Method 1600:Method Time to Detection (h)Method Time to Detection (h)
Rapid Biosensor Detection of Rapid Biosensor Detection of E. E. coli coli O157:H7 in SpinachO157:H7 in Spinach
Biosensor ApplicationsBiosensor ApplicationsBioterrorism/Bioterrorism/biowarfarebiowarfare agents (anthraxagents (anthrax, , plague, smallpox, toxins) (U.S. Army, NIH)plague, smallpox, toxins) (U.S. Army, NIH)Airborne pathogens (post office, sick building Airborne pathogens (post office, sick building syndrome) (USPS)syndrome) (USPS)Waterborne pathogens (Waterborne pathogens (Cryptosporidium, E. Cryptosporidium, E. colicoli, , Salmonella, Salmonella, cholera) (PCU, EPA)cholera) (PCU, EPA)FoodborneFoodborne pathogens (pathogens (E. coli E. coli O157:H7, O157:H7, Salmonella, Salmonella, ListeriaListeria) (FDA, USDA)) (FDA, USDA)Infectious diseases (blood, feces)Infectious diseases (blood, feces)Recovery of live bacteria for viability Recovery of live bacteria for viability confirmation, DNA analysis, and forensic confirmation, DNA analysis, and forensic evidenceevidence
Agencies Supporting Biosensor Research:Agencies Supporting Biosensor Research:
U.S. Army/RDECOMU.S. Army/RDECOMNational Institutes of Health (SBIR)National Institutes of Health (SBIR)National Science Foundation (SBIR, STTR)National Science Foundation (SBIR, STTR)U.S. Environmental Protection AgencyU.S. Environmental Protection AgencyWater Environment Research FoundationWater Environment Research FoundationPinellas County UtilitiesPinellas County UtilitiesSouthern California Water Coastal Water Southern California Water Coastal Water Research ProjectResearch ProjectCooperative Institute for Coastal and Cooperative Institute for Coastal and EstaurineEstaurineEnvironmental Technology (CICEET)Environmental Technology (CICEET)National CattlemenNational Cattlemen’’s Beef Associations Beef Association
Corporate, Government, and University Partners
EPA
Marianne
Joyce Betty
Stephaney
SoniaSarah Kim Alicia
Sonja
www.usf.edu/abl