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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