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COMPOSITE SURFACE FOR CAPTURE OF LISTERIA MONOCYTOGENES FOR

PROTEIN BIOCHIPSTom Huang,1,7 Jennifer Sturgis,2 Rafael Gomez,3 Tao Geng,4 Rashid

Bashir,3,5 Arun K. Bhunia,4 J. Paul Robinson,2,5 and Michael R. Ladisch5,6,7

1School of Chemical Engineering, 2Department of Basic Medical Sciences, 3School of Electrical and Computer Engineering, 4Molecular

Food Microbiology Laboratory, Department of Food Science, 5Department of Biomedical Engineering, 6Department of Agricultural and

Biological Engineering, 7Laboratory of Renewable Resources Engineering

Purdue University, West Lafayette, IN. August 2002

ACKNOWLEDGEMENT•Research supported through a cooperative agreement with ARS of USDA (Project No. 1935-42000-035)

•Richard Linton (FSEC at Purdue University)

•Randy Woodson (ARS at Purdue University)

•Amanda Lathrop, Sang-won Lee

•Tim Miller, Jack Denton, Bill Crabill

•LORRE group members

OUTLINE

Background Information

Materials and Methods

Results and Discussion

Conclusions

LISTERIA MONOCYTOGENES

•Food-born pathogen

•Gram positive (1µm x 2 µm )

•Growth temperature (1-45 ºC)

•Acid and salt tolerant

•Cause listeriosis

•Annual cases >2,500; Mortality 20-28%

CURRENT DETECTION METHODS

•Conventional culture method (5 –7 days)

•Pre-enrichment growth•Selective enrichment culture•Selective diagnostic plating •Biochemical identification

•Enzyme Linked Immunosorbent Assay (3 –5 days)

•Applied after selective enrichment •Using direct ELISA sandwich technique•Or using indirect ELISA sandwich technique•Detects 106 cells per microtiter well

THE NEED

• Sensitivity• Time to result (few hours)• Portable• Easy to use

BIOCHIP

•Microfabricated device

•Rapidly detect and analyze biological species

•Applications in industries such as agriculture, foods, health care

MICROFLUIDIC BIOCHIP

G lass cover

In/O ut ports

C hannels/W ells

E poxy adhesive

Pin

•Closed well microfluidic device

• Fluidic input/output ports

• Sealed channels and wells with electrodes for electronic detection

• Detection through change in the impedance measurement

OBJECTIVE

• Attach antibody on biochip surface

• Block non-specific adsorptions

Cavities with Pt electrodes

20 µm wide channel

Input port

SURFACE MODIFICATION

SiO2Si

OHO

HO

HO

HO

HO

HO

HO

HO

H

octadecyltrichlorosilane Si

Cl

Cl

Cl

SiO O O

SiO O O

SiO O O

SiO O O

SiO2Si

Hydrophilic microchip surface with a contact angle of ~ 2 º

Hydrophobic microchip surface modified with C18 with a contact angle of ~ 110 º

ANTIBODY ATTACHMENT Biotinylated BSA “Blocks and Anchors”

Streptavidin

Biotinylated antibody C11E9~14 nm

~10 nm

~4 nm

Hydrophobic silica surface modified with C18

Biotinylated BSA

* Size information are obtained from Biochemistry 2nd edition, R. H. Garrett and C. M. Grisham, 1995.

EXPERIMENTAL METHODS

•Validating approach•Surface plasmon resonance•Binding of streptavidin to covalently immobilized biotin vs. physically adsorbed biotinylated BSA

•Characterize surfaces •Fluorescence Microscopy•Visualizing adsorptions of proteins, antibody,and bacteria cells on microchip surface

SURFACE PLASMON RESONANCE

Vibro-stirrer

Prism

Sample Well

Resonant MirrorLow R.I. Layer

Coupling Layer

Polarizer

PHYSICALLY ADSORBED BIOTINYLATED BSA VS.COVALENTLY IMMOBILIZED BIOTIN

SURFACE PLASMON RESONANCE RESULTS

• Physically adsorbed biotinylated BSA effectively captures streptavidin

• Streptavidin successfully anchor biotinylated antibody C11E9

• Non-specific adsorption not characterized

CHARACTERIZING MICROCHIP SURFACE

•Microchips for adsorption studies

•PECVD fabricated oxide layer

•SiO2 with Pt patterns

Blank chip

BSA-FITC adsorption

FLUORESCENCE MICROSCOPYSAMPLE ADSORPTION PROTOCOL

•Incubate BSA-FITC with a microchip in a well for 30 minutes

•Washing 3 time to rid of excess

•View under microscope

IntensityMean = 48.19Std. Dev = 0.51

IntensityMean = 243.81Std. Dev = 0.93

BSA ADSORPTIONS

82

255

50

39

237

0 50 100 150 200 250 300

Fluoresence intensity

Optical saturation a. Blank (chip only, no

BSA) b. Hydrophilic surface,

pH 7.2 c. Hydrophobic C18

surface, pH 7.2 d. Hydrophilic surface,

pH 4.9 e. Step d followed by

washing with pH 7.2 buffer

STREPTAVIDIN AND BIOTIN BINDING

140

31

255

0 50 100 150 200 250 300

Fluoresence intensity

a. Blank (chip only, no protein or biotin)

b. Streptavidin on

biotinylated BSA c. Biotin on streptavidin

Optical saturation

ADSORPTION OF NON-BIOTINYLATED IgG TO VAIOUS SURFACES

255

31

31

34

0 50 100 150 200 250 300

Fluoresence intensity

a. Blank (chip only, no protein) b. Biotinylated BSA c. Streptavidin d. Hydrophobic C18

Optical saturation

FLUORESENCE IMAGES OF BACTERIAL ADSORPTION

BSA Surface B

DC BSA Surface

Hydrophobic C18 Surface

Hydrophobic C18 Surface

E. coli

Listeria monocytogenes

E. coli

Listeria monocytogenes

ADSORPTION OF E. COLI (~108 cells/ml in PBS) TO VARIOUS SURFACES

9

1

1

25

0 20 40 60 80 100 120

Average number of cells per 435um x 435um area

a. BSA b. Biotinylated BSA c. Streptavidin d. Streptavidin

blocked by BSA e. Hydrophobic C18

>100

ADSORPTION OF LISTERIA MONOCYTOGENES (~107 cell/ml in PBS) TO VARIOUS SURFACES

>100

60

45

20

2

0 20 40 60 80 100 120

Average number of cells per 435um x 435um area

a. Biotinylated BSA b. Streptavidin c. Streptavidin

blocked by BSA d. Biotinylated

antibody (C11E9) e. Hydrophobic C18

CONCLUSIONS

• Biotinylated BSA “Blocks and Anchors”• Physically adsorbs onto hydrophobic C18 surface

• Blocks nonspecific adsorption

• Anchors IgG in biotin-streptavidin sandwich

• Contact time is short for protein adsorption• Biotinylated BSA to C18 surface(~30 minutes)

• Streptavidin to biotinylated BSA (<2 minutes)

• Biotinylated antibody C11E9 to streptavidin (~10 minutes)

• L. monocytogenes cells successfully captured

STOPS HERE !

FLUORESCEIN ISOTHIOCYANATE•Also known as FITC

•Fluorescence dye for labeling proteins, bacteria, etc.

•Reacts with amino, sulfhydryl, imidzaoyl, tyrosyl and carbonyl groups

For example:R1N=C=S (FITC) + R2NH2 (Primary amine) R1NH-C-NHR2 (Thiourea)

•Excitation wavelength 495 nm

•Emission wavelength 520 nm

•520 nm corresponds to green color

PROTEIN/ANTIBODY IMMOBILIZATION THROUGH COVALENT ATTACHMENT

Aminosilane NH2 NH2 NH2 NH2 NH2

Silica surface

OH OH OH OH

Amino surface

Glutaraldehyde

O

O OH HH2N – IgG or proteins

NH

O

O O

H

H H

HHN-IgG

Immobilized IgG antibody or proteins

NH2

Glutaraldehyde coated surface

PROTEIN/ANTIBODY IMMOBILIZATION THROUGH PHYSICAL ADSORPTION

Hydrophilic silica with native negative charge

Lateral protein to protein electrostatic interaction

Negatively charged protein at pH 7.2

Protein to surface electrostaticinteraction

Hydrophobic silica surface modified with C-18 end groups

Hydrophobic portion of a protein

Hydrophobic interaction might distort the native state of a protein Protein to

surface hydrophobic interaction

ELECTRICAL CHARGES ON A PROTEIN AND SILICA SURFACE

IsoelectricpH+

543 6 72 8Net charge of protein

0

-pH of a protein such as BSA

Negatively charged at pH 7.2OH OH OH OH OH OH OH

Hydrophilic silica with native negative charge

CH3 CH3 CH3 CH3 CH3 CH3 CH3 Neutral at pH 7.2

Hydrophobic silica surface modified with C-18 end groups

ANTIBODY

Fc

Fab Fab

SS

SS

SS

SS

SS

SSS

SS

S

SS

SS

SS

SS

N NNN

Fc

Fab Fab

Antigen binding sites

Disulfide bond

Antigen

Surface of a biochip

* 3-D IgG molecule is obtained from David Wild (http://www.techfak.uni-bielefeld.de/bcd/ForAll/Introd/antibody.html).

Antibody antigen interaction

NON-SPECIFIC BINDING OF ANTIGEN

Streptavidin

Biotinylated antibody c11e9

Hydrophobic silica surface modified with C-18

Antigen Specific binding

Non-specific binding

Biotinylated BSA

NON-SPECIFIC BINDING OF ANTIBODY

Specific bindingNon-specific binding

Streptavidin

Hydrophobic silica surface modified with C-18

Biotinylated antibody c11e9

Biotinylated BSA

ANTIBODY ORIENTATIONIncorrectly oriented antibodyCorrectly oriented antibody

Antigen bindingsite

Fc unit

Surface

IgG class antibody

Protein G Streptavidin

Biotinylated antibody

Protein A

Surface

STREPTAVIDIN-BIOTIN INTERACTION

•Each streptavidin binds 4 units of biotin

•Strongest known noncovalent, biological recognition (Ka=1015 M-1)

•Bond formation is rapid and extremely stable

•Can withstand up to 3 M guanidine HCl

•Can be released by 8 M guanidine HCl at pH 1.5, or by autoclaving

Streptavidin

Biotin

PROTEINS•Bovine Serum Albumin (BSA)

•Models adsorption of biotinylated BSA•66,000 daltons•Isoelectric pH of 4.7-4.8•Biotinylated BSA has 8 moles biotin per mole of BSA

•Streptavidin•Isolated from Streptomycetes•60,000 daltons•Isoelectric pH of 5.0•Lower non-specific binding compared to avidin

•Biotin•A vitamin found in tissues and blood•244 daltons•Binds with high affinity to streptavidin

ANTIBODY AND LISTERIAINNOCUA

•Antibody c11e9•150,000 daltons IgG•Binds to Listeria monocytogenes and Listeria innocua•Biotinylated using instruction from Pierce

•Anti-mouse IgG•Binds to antibody c11e9•Used for binding studies in surface plasmon resonance and non-specific binding studies in fluorescence microscopy

•Listeria innocua•Non-pathogenic•Models adsorption of Listeria monocytogenes

RECOMMENDATION• NeutrAvidinTM can be used as a substitute for

streptavidin

• IAsys cuvette with hydrophobic surface can be used to obtaining constants such as, kass, kdiss, and Ka

• Determining amount of proteins adsorbed at surface, and protein film thickness using ellipsometrytechnique

• Characterizing the surface of protein film using atomic force microscopy (AFM)

• New attachment scheme can be tried by adsorb the NeutrAvidinTM onto the gold surface pre-immobilized with biotinylated DNA