BIO-MEDIATED ASSEMBLY OF FUNCTIONALIZED MICROBEADS FOR CAPTURE OF MICROORGANISMS

Transcription

1 BIO-MEDIATED ASSEMBLY OF FUNCTIONALIZED MICROBEADS FOR CAPTURE OF MICROORGANISMS Tom Huang, 1,7 Tao Geng, 2 Demir Akin, 3 Woo-Jin Chang, 3 Jennifer Sturgis, 4 Rashid Bashir, 3,5 Arun K. Bhunia, 2 J. Paul Robinson, 4,5 and Michael R. Ladisch 5,6,7 1 School of Chemical Engineering, 2 Molecular Food Microbiology Laboratory, Department of Food Science, 3 Department of Biomedical Engineering, 4 Department of Basic Medical Sciences, 5 School of Electrical and Computer 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 ARS of USDA (Project No ) Richard Linton (FSEC at Purdue University) Randy Woodson (ARS at Purdue University) Tim Miller, C-Y Fong, Bill Crabill LORRE group members

3 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%

4 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 10 6 cells per microtiter well

5 OBJECTIVE Cavities with Pt electrodes Input port 20 µm wide channel Attach antibody on microchip surface Selectively capture targeted organism Block non-specific adsorptions

6 SURFACE MODIFICATION O H O Octadecyltrichlorosilane (OTS) H O H O H O SiO 2 Si Si H O H O H O H O H Cl Si Cl Cl Hydrophilic microchip surface with a contact angle of ~ 2 º Si Si Si Si O O OO O OO O OO O O SiO 2 Hydrophobic microchip surface modified with C18 with a contact angle of ~ 110 º Si * Ladisch MR Bioseparations Engineering. New York: John Wiley & Sons. p

7 PREVIOUS WORK Biotinylated BSA Blocks and Anchors ~14 nm Biotinylated antibody C11E9 ~10 nm Streptavidin ~4 nm Biotinylated BSA Hydrophobic silica surface modified with C18 * Huang et al., Composite surface for blocking bacterial adsorptions Biotechnology and Bioengineering, 81:

8 PHYSICALLY ADSORBED BIOTINYLATED BSA VS. COVALENTLY IMMOBILIZED BIOTIN

9 FLUORESENCE IMAGES OF BACTERIAL ADSORPTION B E. coli E. coli BSA Surface Hydrophobic C18 Surface C D Listeria monocytogenes Listeria monocytogene BSA Surface Hydrophobic C18 Surface

10 MICRO-ASSEMBLY OF MICROBEADS THROUGH BIOTIN-STREPTAVIDIN ~14 nm Biotinylated antibody C11E9 ~2 µm Streptavidin coated microbeads (2.8 µm) ~4 nm Biotinylated BSA Hydrophobic silica surface modified with C18 * Drawing not to scale.

11 MICRO-ASSEMBLY OF MICROBEADS THROUGH BSA ~0.8 µm ~4 nm Dimethylamino or polystyrene microbeads (0.8 µm) BSA Hydrophobic silica surface modified with C18 * Drawing not to scale.

12 ADVANTAGE OF BEADS-BASED SURFACES Commercially available Sizes ranging from 10 nm-3 µm Various types of chemistry Anchors onto surface thorough surface chemistry Easily identifiable in microfluidic chips Provides increased surface area Various shapes Controlled porosities

13 EXPERIMENTAL INFORMATION Fluidic self-assembly Microbeads concentration ~10^5-10^6 particle/ml PBS buffer ph 7.4 Protein coated microchip

14 STREPTAVIDIN BEADS ADSORPTION ON BIOTINYLATED BSA SURFACE

15 ANTI-LISTERIA ANTIBODY COATED BEADS ADSORPTION ON C18 SURFACE

16 TIME COURSE STUDY ON MICROBEADS ADSORPTION 80 Beads coverage (10^3/mm^2) Streptavidin beads adsorption on biotinylated BSA surface Anti-Listeria beads adsorption on C18 surface Time (hr)

17 DIMETHYLAMINO BEADS ADSORPTION ON BSA SURFACE Dimethylamino beads Dimethylamino beads

18 POLYSTYRENE BEADS ADSORPTION ON BSA SURFACE Polystyrene beads Polystyrene beads

19 VALIDATION OF BIOTINYLATED ANTI-LISTERIA ANTIBODY ATTACHMENT TO STREPTAVIDIN BEADS Streptavidin beads with biotinylated anti- Listeria antibody Anti-mouse IgG adsorption to streptavidin beads with biotinylated anti- Listeria antibody * 30 minutes incubation with Anti-mouse IgG ( 50 µg/ml) in PBS buffer ph 7.4 at room temperature.

20 BACTERIAL ADSORPTION ON STREPTAVIDIN BEADS COATED WITH BIOTINYLATED ANTI- LISTERIA ANTIBODY * 60 minutes incubation with bacterial cells (10^7-10^8 cells/ml) in PBS buffer ph 7.4 at room temperature.

21 BACTERIAL ADSORPTION ON DIMETHYLAMINO BEADS * 60 minutes incubation with bacterial cells (10^7-10^8 cells/ml) in PBS buffer ph 7.4 at room temperature.

22 BACTERIAL ADSORPTION ON POLYSTYRENE BEADS * 60 minutes incubation with bacterial cells (10^7-10^8 cells/ml) in PBS buffer ph 7.4 at room temperature.

23 VALIDATION OF BACTERIAL CAPTURE BY BEADS Streptavidin beads with biotinylated anti- Listeria antibody Anti-Listeria antibody coated beads Polystyrene beads Dimethylamino beads

24 NUMBER OF BACTERIAL CELLS ADSORBED ON VARIOUS SURFACES IN A 435 µm X 435 µm AREA Microbead-coated surface: E. coli Listeria monocytogenes Streptavidin beads with biotinylated anti- Listeria antibody Anti-Listeria beads blocked with BSA Polystyrene beads Dimethylamino beads Protein-coated surface: * Biotinylated BSA 1 2 Streptavidin Streptavidin blocked by BSA 9 20 Streptavidin with biotinylated anti- Listeria antibody No data *Data obtained from Biotechnology & Bioengineering, (Huang et al., 2003). 60

25 CONCLUSIONS Biotinylated BSA/BSA Immobilizes streptavidin beads through biotin Immobilizes polystyrene and dimethylamino beads through hydrophobic or electrostatic adsorptions Streptavidin beads anchors biotinylated IgG Block non-specific adsorptions Microbeads-based surface Forms highly packed monolayer Functionalized beads for specific capture Produces higher capture than protein-based surface L. monocytogenes cells successfully captured

26 STOP!!!

27 MICROFLUIDIC BIOCHIP Pin Glass cover Channels/W ells In/Out ports Increased sensitivity Rapid detection Closed well microfluidic device Fluidic input/output ports Sealed channels and wells with electrodes for electronic detection Epoxy adhesive