Microfluidic Devices that Capture Bacteria for Growth and Kill Analysis

Transcription

1 Microfluidic Devices that Capture Bacteria for Growth and Kill Analysis Michael J. Lochhead, PhD. Accelr8 Technology Corporation Denver, CO AVS Symposium Biomaterial Interfaces Microbe-Surface Interactions November 14, 2006

2 Microbe-Surface Interactions in a Clinical Diagnostics Context: Rapid Bacterial Detection and Antibiotic Susceptibility Testing Hospital-acquired pneumonia ICU s #1 infectious killer >300,000 cases/yr US only ~40,000 to 90,000 avoidable deaths 14-November-2006 Accelr8 Technology Corp. 2

3 Analysis of Infectious Bacteria is still Culture-Based Days to Result CULTURE = same basic strategy as in the 1870s 1970s: mechanization, still using cultures Typically 24 to 72 hours to result Physician starts empiric therapy long before lab results available Incorrect initial therapy yields poor patient outcomes Contributes to emerging antibiotic resistant organisms 14-November-2006 Accelr8 Technology Corp. 3

4 Instrument Development Strategy Combine traditional microbiology techniques in a novel, automated device format Microscopy-based analysis Label-free (darkfield) microscopy Fluorescence microscopy Automated image analysis algorithms and database management Disposable fluidic cassette 14-November-2006 Accelr8 Technology Corp. 4

5 Single-Use Fluidic Cassettes Multiple technologies satisfy different functions Injection molded plastic body Sample and reagent reservoirs (pipet loading) Waste Manifold interface to pump station Laminated fluidic layers (channel dimensions ~ 500 um) Flowcells (8) with capture surface and for imaging windows 14-November-2006 Accelr8 Technology Corp. 5

6 5-Step Assay SPECIMEN (off cassette prep) Bronchoalveolar Lavage (BAL) Load cassette ON-CASSETTE CAPTURE IDENTIFY GROW KILL IDCount < 2 hours Antibiotic Panel Profile (AST) < 8 hours 14-November-2006 Accelr8 Technology Corp. 6

7 Assay Details: Sample Chamber Schematic Transparent Electrodes Polymer Coating ~ 400 um Bacterial Sample Off-line prep Suspended in proprietary buffer Hydrodynamic (pressure-driven) flow through flow channels 14-November-2006 Accelr8 Technology Corp. 7

8 Assay Details: Electrokinetic Concentration Apply Low Voltage (< 2V) Redox reactions create E field Electrokinetic concentration to capture surface Polymer coating facilitates adhesion 14-November-2006 Accelr8 Technology Corp. 8

9 Assay Details: Bacterial Count and Identification Microscope Objective Real-time image acquisition throughout process 14-November-2006 Accelr8 Technology Corp. 9

10 Assay Details: Bacterial Count and Identification Fluorescent Reagent Cocktail Antibodies Gram stain Mortal stain On-board incubation and programmed washes 14-November-2006 Accelr8 Technology Corp. 10

11 Assay Details: Bacterial Growth Monitor growth of individual clones Generate quantitative growth curves 14-November-2006 Accelr8 Technology Corp. 11

12 Assay Details: Antibiotic Susceptibility Testing Antibiotic Flow in antibiotic Monitor drug efficacy Change in growth kinetics Mortal stain signal 14-November-2006 Accelr8 Technology Corp. 12

13 Electrokinetic Capture CAPTURE Elapsed Time < 5 minutes Microscope Objective TIME-LAPSE PHOTOS Darkfield Microscopy (false color images) Elapsed time < 2 minutes Bug size ~ 1 5 microns; 20X objective 14-November-2006 Accelr8 Technology Corp. 13

14 Species Identification Elapsed Time CAPTURE < 5 minutes IDENTIFY 30 minutes Label with antibodies against each species. Antibodies have fluorescent tags of different colors. 14-November-2006 Accelr8 Technology Corp. 14

15 Map and Count Elapsed Time CAPTURE < 5 minutes IDENTIFY 30 minutes Assign a physical address to each individual bacterium. Enables analysis of the same individuals over time. Quantum Microbiology TM 14-November-2006 Accelr8 Technology Corp. 15

16 Growth Measurement CAPTURE IDENTIFY GROW Elapsed Time < 5 minutes 30 minutes minutes 1.E08 Integrated Intensity 1.E07 1.E06 1.E E Integrated Intensity t (minutes) 1.E06 Growth curves 6 clone example 117 y = e0.1035x R 2 = E t (minutes Ensemble average 14-November-2006 Accelr8 Technology Corp. 16

17 Antibiotic Kill Kinetics CAPTURE IDENTIFY GROW KILL Elapsed Time < 5 minutes 30 minutes minutes 2-5 hours Introduce 80 antibiotic and 70 fluorescent mortal stain. Non Stained Clones 60 Count 50 individual killed cells over 40 time. 30 Plot 20 the time-kill curves. 10 Report strain identity and 0 drug action Time (min.) 14-November-2006 Accelr8 Technology Corp. 17

18 Single Clone: Growth and Kill Growth rate Cell count Time of cell kill k 0 = 2.6/hr N = 2 N 1 = min. N 2 = min. 14-November-2006 Accelr8 Technology Corp. 18

19 Accelr8 Rapid Bacterial Detection System Summary Whole organism identification and antibiotic susceptibility testing Bench-top, laboratory instrument Traditional microbiology techniques Disposable fluidic cassette Advanced surface chemistry 14-November-2006 Accelr8 Technology Corp. 19

20 Microbe-Surface Interactions: Diagnostic Device Context Prevent adhesion to assay surfaces in some places Promote adhesion to assay surfaces in others Maintain viability for AST applications Relatively short exposure times (hours) 14-November-2006 Accelr8 Technology Corp. 20

21 OptiChem Surface Chemistry U.S. Patents 6,844,028 and 7,067,194 Poly (ethylene glycol) PEG-based technology Multicomponent, cross-linked formulation Thin hydrogel architecture Readily functionalized Substrate 14-November-2006 Accelr8 Technology Corp. 21

22 OptiChem Surface Chemistry Engineered for low non-specific binding Activated functional groups (e.g. amine-reactive groups) Multicomponent Polymer: 3D, permeable matrix Low non-specific binding Optimum surface energy Substrate Attachment to multiple substrate materials (glass, plastics, metal oxides) 14-November-2006 Accelr8 Technology Corp. 22

23 OptiChem Coating Architecture: AFM surface topography (under water, tapping mode) DR Y Dry Thickness: ~ 10 nm (ellipsometry) UNDERWATER Z-Axis (nm) min. underwater 20 min underwater min underwater Hydrated Thickness: 50 to 100 nm Distance (um) 14-November-2006 Accelr8 Technology Corp. 23

24 Bacterial Retention Assay Gravity settle for one hour to polymer surface Microscope Objective 14-November-2006 Accelr8 Technology Corp. 24

25 Bacterial Retention Microscope Objective Controlled shear-rate washes S. aureus K. pneumoniae % Retention 120% 100% 80% 60% 40% Bare Glass 20% OptiChem 0% Wall Shear Rate (sec-1) 120% 100% 80% 60% 40% 20% 0% Bare Glass OptiChem Wall Shear Rate (sec-1) 14-November-2006 Accelr8 Technology Corp. 25 % Retention

26 OptiChem Functionalization Functional groups Commercialized for Microarray Apps: N-hydroxy succinimide (NHS) Streptavidin Substrate 14-November-2006 Accelr8 Technology Corp. 26

27 OptiChem Functionalization Functional groups Functionalize for Bacterial Capture: Specific: antibodies, lectins, etc. Non-Specific: polycations Substrate 14-November-2006 Accelr8 Technology Corp. 27

28 OptiChem Functionalization Microarray, Whole Organism, Sandwich Immunoassay 1. Print array of specific monoclonal antibodies 2. Sample: Mixed E. coli S. aureus 3. Mixed primary antibodies 4. Mixed detection molecules Location control row (Rabbit IgG) Capture: G anti E. coli O157:H7 Primary: R anti E. coli Detection: G anti Rabbit A555 Capture: M anti S. aureus (mab) Primary: M anti S. aureus, biotin Detection: Streptavidin-A647 Mixed capture Location control row (Rabbit IgG) 14-November-2006 Accelr8 Technology Corp. 28

29 OptiChem Functionalization Non-Specific Capture of Whole Organisms Cationic OptiChem Concepts: Polypeptides (e.g., poly-l-lysine) Amine functionality Basic (cationic) proteins Substrate 14-November-2006 Accelr8 Technology Corp. 29

30 OptiChem Surface Chemistry Multicomponent, cross-linked, PEG-based films Outstanding inhibition of protein and bacterial cell adhesion Readily functionalized for specific attachment Scalable coating technology Acknowledgements: Chad Greef Steve Metzger Dave Howson Ken Emoto Jim Mascali Misty Lindsey Nisha Kulpathipranja Mike Lochhead 14-November-2006 Accelr8 Technology Corp. 30