COMPOSITE SURFACE FOR CAPTURE OF LISTERIA MONOCYTOGENES FOR PROTEIN BIOCHIPS

Transcription

1 COMPOITE URFACE FOR CAPTURE OF LITERIA MONOCYTOGENE FOR PROTEIN BIOCHIP Tom Huang, 1,7 Jennifer turgis, 2 Rafael Gomez, 3 Tao Geng, 4 Rashid Bashir, 3,5 Arun K. Bhunia, 4 J. Paul Robinson, 2,5 and Michael R. Ladisch 5,6,7 1 chool of Chemical Engineering, 2 Department of Basic Medical ciences, 3 chool of Electrical and Computer Engineering, 4 Molecular Food Microbiology Laboratory, Department of Food cience, 5 Department of Biomedical Engineering, 6 Department of Agricultural and Biological Engineering, 7 Laboratory of Renewable Resources Engineering Purdue University, West Lafayette, IN. August 2002

2 ACKNOWLEDGEMENT Research supported through a cooperative agreement with AR of UDA (Project No ) Richard Linton (FEC at Purdue University) Randy Woodson (AR at Purdue University) Amanda Lathrop, ang-won Lee Tim Miller, Jack Denton, Bill Crabill LORRE group members

3 OUTLINE Background Information Materials and Methods Results and Discussion Conclusions

4 LITERIA MONOCYTOGENE 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%

5 CURRENT DETECTION METHOD Conventional culture method (5 7 days) Pre-enrichment growth elective enrichment culture elective diagnostic plating Biochemical identification Enzyme Linked Immunosorbent Assay (3 5 days) Applied after selective enrichment Using direct ELIA sandwich technique Or using indirect ELIA sandwich technique Detects 10 6 cells per microtiter well

6 THE NEED ensitivity Time to result (few hours) Portable Easy to use

7 BIOCHIP Microfabricated device Rapidly detect and analyze biological species Applications in industries such as agriculture, foods, health care

8 MICROFLUIDIC BIOCHIP Glass cover Channels/W ells Closed well microfluidic device Pin In/Out ports Fluidic input/output ports ealed channels and wells with electrodes for electronic detection Detection through change in the impedance measurement Epoxy adhesive

9 OBJECTIVE Cavities with Pt electrodes 20 µm wide channel Attach antibody on biochip surface Input port Block non-specific adsorptions

10 URFACE MODIFICATION O H O octadecyltrichlorosilane H O H O H O io 2 i H O H O H O H O H Cl i Cl Hydrophilic microchip surface with a contact angle of ~ 2 º Cl i i i i O O OO O OO O OO O O io 2 i Hydrophobic microchip surface modified with C18 with a contact angle of ~ 110 º

11 ANTIBODY ATTACHMENT Biotinylated BA Blocks and Anchors ~14 nm Biotinylated antibody C11E9 ~10 nm treptavidin ~4 nm Biotinylated BA Hydrophobic silica surface modified with C18 * ize information are obtained from Biochemistry 2 nd edition, R. H. Garrett and C. M. Grisham, 1995.

12 EXPERIMENTAL METHOD Validating approach urface plasmon resonance Binding of streptavidin to covalently immobilized biotin vs. physically adsorbed biotinylated BA Characterize surfaces Fluorescence Microscopy Visualizing adsorptions of proteins, antibody, and bacteria cells on microchip surface

13 URFACE PLAMON REONANCE Vibro-stirrer ample Well Prism Coupling Layer Resonant Mirror Low R.I. Layer Polarizer

14 PHYICALLY ADORBED BIOTINYLATED BA V. COVALENTLY IMMOBILIZED BIOTIN

15 URFACE PLAMON REONANCE REULT Physically adsorbed biotinylated BA effectively captures streptavidin treptavidin successfully anchor biotinylated antibody C11E9 Non-specific adsorption not characterized

16 CHARACTERIZING MICROCHIP URFACE Microchips for adsorption studies PECVD fabricated oxide layer io 2 with Pt patterns

17 FLUORECENCE MICROCOPY AMPLE ADORPTION PROTOCOL Incubate BA-FITC with a microchip in a well for 30 minutes Washing 3 time to rid of excess View under microscope Blank chip Intensity Mean = td. Dev = 0.51 BA-FITC adsorption Intensity Mean = td. Dev = 0.93

18 BA ADORPTION Fluoresence intensity a. Blank (chip only, no BA) 39 Optical saturation b. Hydrophilic surface, ph c. Hydrophobic C18 surface, ph d. Hydrophilic surface, ph e. tep d followed by washing with ph 7.2 buffer 82

19 TREPTAVIDIN AND BIOTIN BINDING Fluoresence intensity a. Blank (chip only, no protein or biotin) 31 Optical saturation b. treptavidin on biotinylated BA 255 c. Biotin on streptavidin 140

20 ADORPTION OF NON-BIOTINYLATED IgG TO VAIOU URFACE Fluoresence intensity a. Blank (chip only, no protein) 31 Optical saturation b. Biotinylated BA 34 c. treptavidin 31 d. Hydrophobic C18 255

21 FLUOREENCE IMAGE OF BACTERIAL ADORPTION BA urface B Hydrophobic C18 urface E. coli E. coli C BA urface D Hydrophobic C18 urface Listeria monocytogenes Listeria monocytogenes

22 ADORPTION OF E. COLI (~10 8 cells/ml in PB) TO VARIOU URFACE Average number of cells per 435um x 435um area a. BA 1 b. Biotinylated BA 1 c. treptavidin 25 d. treptavidin blocked by BA 9 e. Hydrophobic C18 >100

23 ADORPTION OF LITERIA MONOCYTOGENE (~10 7 cell/ml in PB) TO VARIOU URFACE Average number of cells per 435um x 435um area a. Biotinylated BA 2 b. treptavidin 45 c. treptavidin blocked by BA 20 d. Biotinylated antibody (C11E9) 60 e. Hydrophobic C18 >100

24 CONCLUION Biotinylated BA 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 BA to C18 surface(~30 minutes) treptavidin to biotinylated BA (<2 minutes) Biotinylated antibody C11E9 to streptavidin (~10 minutes) L. monocytogenes cells successfully captured

25 TOP HERE!

26 FLUORECEIN IOTHIOCYANATE For example: Also known as FITC Fluorescence dye for labeling proteins, bacteria, etc. Reacts with amino, sulfhydryl, imidzaoyl, tyrosyl and carbonyl groups R 1 N=C= (FITC) + R 2 NH 2 (Primary amine) R 1 NH-C-NHR 2 (Thiourea) Excitation wavelength 495 nm Emission wavelength 520 nm 520 nm corresponds to green color

27 PROTEIN/ANTIBODY IMMOBILIZATION THROUGH COVALENT ATTACHMENT OH OH OH OH ilica surface Aminosilane NH 2 NH 2 NH 2 NH 2 NH 2 Amino surface Glutaraldehyde H O H O H 2 N IgG or proteins H O H O O O H NH HN-IgG H NH 2 Immobilized IgG antibody or proteins Glutaraldehyde coated surface

28 PROTEIN/ANTIBODY IMMOBILIZATION THROUGH PHYICAL ADORPTION Negatively charged protein at ph 7.2 Lateral protein to protein electrostatic interaction Protein to surface electrostatic interaction Hydrophilic silica with native negative charge Hydrophobic portion of a protein Hydrophobic interaction might distort the native state of a protein Protein to surface hydrophobic interaction Hydrophobic silica surface modified with C-18 end groups

29 ELECTRICAL CHARGE ON A PROTEIN AND ILICA URFACE + Isoelectric ph Net charge of protein ph of a protein such as BA OH OH OH OH OH OH OH Negatively charged at ph 7.2 Hydrophilic silica with native negative charge CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 Neutral at ph 7.2 Hydrophobic silica surface modified with C-18 end groups

30 ANTIBODY Antigen binding sites F ab F ab N N F ab F ab N N F c F c Disulfide bond Antigen Antibody antigen interaction urface of a biochip * 3-D IgG molecule is obtained from David Wild (

31 NON-PECIFIC BINDING OF ANTIGEN Antigen pecific binding Non-specific binding Biotinylated antibody c11e9 treptavidin Biotinylated BA Hydrophobic silica surface modified with C-18

32 NON-PECIFIC BINDING OF ANTIBODY Non-specific binding pecific binding Biotinylated antibody c11e9 treptavidin Biotinylated BA Hydrophobic silica surface modified with C-18

33 ANTIBODY ORIENTATION Correctly oriented antibody Incorrectly oriented antibody Antigen binding site Fc unit urface IgG class antibody Protein A Protein G Biotinylated antibody treptavidin urface

34 TREPTAVIDIN-BIOTIN INTERACTION Each streptavidin binds 4 units of biotin treptavidin trongest known noncovalent, biological recognition (K a =10 15 M -1) Bond formation is rapid and extremely stable Biotin Can withstand up to 3 M guanidine HCl Can be released by 8 M guanidine HCl at ph 1.5, or by autoclaving

35 PROTEIN Bovine erum Albumin (BA) Models adsorption of biotinylated BA 66,000 daltons Isoelectric ph of Biotinylated BA has 8 moles biotin per mole of BA treptavidin Isolated from treptomycetes 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

36 ANTIBODY AND LITERIA INNOCUA 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

37 RECOMMENDATION NeutrAvidin TM can be used as a substitute for streptavidin IAsys cuvette with hydrophobic surface can be used to obtaining constants such as, k ass, k diss, and K a Determining amount of proteins adsorbed at surface, and protein film thickness using ellipsometry technique Characterizing the surface of protein film using atomic force microscopy (AFM) New attachment scheme can be tried by adsorb the NeutrAvidin TM onto the gold surface pre-immobilized with biotinylated DNA