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