Integration of Optical Biosensor Systems for Point of Use Frances S. Ligler, D.Phil., D.Sc. Naval Research Laboratory Washington, DC Movement to Point of Use: Drivers I. Public concerns II. Immediacy: Eliminate sample transportation III. New concepts for molecular recognition IV. Integration of microfluidics and optics/electronics V. Simplified fabrication technologies VI. Miniaturization in electronics & communication VII. Systems integration choices I. Public Concerns* • • • • • • Medical awareness—point of care Diagnostics for developing countries Homeland security Clean drinking water Other toxic materials, nanotechnology ―threat‖ Energy USER DRIVES SYSTEM DESIGN *Grant funding/market opportunity NRL Automated Biosensors 1996-2012 Lifepoint Impact Fast 2000 Analyte 2000 on UAV BioHawk Fast 6000 RAPTOR Plus CT-Array Biosensor mBio Leopard Signalyte UUV mounted immunosensor II. Immediacy: Eliminate sample transportation • Medical – Hospital bedside – Doctor’s office – Home – Remote telemedicine • Environmental monitoring: pollution & climate change • Food safety from source to store • Homeland security and military operations Autonomous Mobile Biosensor Systems Spinoffs: Portable cyclone air samplers and fiber optic biosensors (Research International) Robust air sampler for BioWatch Future: Swarms •Remote identification •10 lb payload •Ram air cyclone •4-fiber biosensor •Lyophilized antibodies •Assays at 5 min intervals • Real-time data transfer •Successful demos at Dugway Proving Ground •Widespread sensing and data collection, agile networks •Nodes can be cell phones, autonomous vehicles, medical devices, other data collection platforms •Changes in chemistry, physiology, epidemiology, safety •Geospatial information from meters to continents Ligler et al. (1998) Remote sensing using an airborne biosensor. Environ. Sci. & Tech., 32, 2461-2466. Microarray Immunosensors Sandwich Concept 2005-2012 Commercial Products Glass slide CT-Array Biosensor NRL Developmental Prototype • • • • • mBio Leopard Small camera 0.4 ft3 box <10 lb. External computer control Moldable reservoirs Golden et al. (2005) Talanta 65, 1078-1085. Golden et al., 1999, SPIE 3602, 132 Golden et al., 1999, SPIE 3602, 132 Microarray Biosensor for the Future IBM concept for $1 multiplexed POC immunosensor for whole blood Published by Gervais et al. in Adv. Mater. 2011, 23, H151–H176 III. New concepts for molecular recognition and signal generation • • • • Single domain antibodies Anti-microbial peptides Carbohydrates Designer peptides Single-domain Antibodies & Fragments 27KDa Human / Rabbit / Mouse 12 & 15 KDa • SdAb are stable: refold after exposure – Heat as high as 95oC – Solvent • Papers by Ellen Goldman & George Anderson Llama SdAb’s heated to 95o, Cooled to 25o Tested for binding to SEB Antimicrobial Peptides for Semi-selective Recognition • • • • Naturally occurring peptides - innate immune system 12-45 amino acids Disrupt microbial membranes + PE PN Ab - PA Ab Semi-selective binding MI CA PB + 0 ng/ml 1 10 100 500 1000 – ID based on pattern of binding Normalized percent of binding 120 E. coli Salmonella Coxiella Brucella 100 80 BotA assays 60 40 •First demo - toxin detection •Environmentally stable 20 0 Magainin Polymyxin B Cecropin A Parasin Bactenicin Polymyxin E Kulagina, et al. (2005) Anal.Chem. 77, 6504-6508; (2006) Anal. Chim. Acta 575, 9-15; (2006) Sens. Act. B, 121, 150-157; (2007) Sensors 7, 2808-2824. Carbohydrate-Target Interactions Bacterium Virus Cell Toxin Glycoprotein Nathan Sharon & Halina Lis in SCIENTIFIC AMERICAN January 1993, 82-89 More Sugars Anti-CT mAb PBS Galβ1-3GalNAcβ β-GalNAc Galβ1-4Glc β Carbohydrate Binding of Toxins • α-Neu5Ac • Neu5Acα2-8Neu5Acα • Neu5Acα2-3Galα1-4Glcβ Campylobacter TT (20) TT (0) • Neu5Acα1-3Galα1-3GalNAcα No recognition Ricin (10) Ricin (5) Ricin (0) Other targets CT (5) CT (0.5) CT (0) •Bot. toxoid A Cy5-CT (5) Cy5-CT (0.5) Cy5-CT (0) • Campylobacter (µg/ml) • Cy5-TT • SEB • E. coli 0157:H7 • Salmonella No recognition Ngundi et al. (2006) Biosens. Bioelectron., 22, 124-130; (2007) Sensor Letts. 5, 621-624. Designer Peptides David Baker’s FoldIt game harnesses human creativity through gaming and social networking to address molecular design problems. Eiben, C. B., et al. Increased Diels-Alderase activity through backbone remodeling guided by Foldit players Nature biotechnology. 30(2), 190-2. (2012) IV. Integration of microfluidics with optics/electronics •Single structure provides multiple functions •Optical and fluidic components integrated into single substrate •Low energy electronic components for data acquisition and transmission •Compatibilty with personal electronics Integrating fluidic channel, sensor surface, and waveguide for signal collection *********** YYYYYYYYYYYYY Excitation Emission * * * * * * * * * * * YYYYYYYYYYYYY Ligler et al. (2002) Anal. Chem.; 74, 713-719. Signalyte CreatvMicrotech Fluid Focusing using Laminar Streams Concept of “Soft Boundaries” Fluids focus fluids 2A) 3A) Fluids focus light. 2B) 3B) 3C) Channel wall Symmetry plane at center of channel Howell et al. (2008) Lab Chip, 8, 1097 - 1103. Hydrodynamic Focusing of Conductance Fluidic Channel Current Electrode Sense Electrodes Permanent Magnets Captured 5µ Beads Current Electrode Inlet 2 Low -Conductivity Confinement Flow Cells Specifically Bound t1 Ionic Buffer R Before V After Outlet Polymer Optics Integrated with Microfluidics Parallel processing without walls Polymer diode array Integrated system focused Wojciechowski, et al. (2009) Organic Photodiodes for Biosensor Miniaturization, Anal.Chem. 81, 3455-3461. Integrated Laser Diodes as Light Sources UV transparent ribbon carrying bare die LED’s Bare die LED being transferred Micromachined trench UV absorbent material ~100 mm Transferred bare die LED Substrate LDW of Blue LED ~ 250 µm Bare LED Metallic Interconnects Connected LED Laser transfer and embedding can also be used for edge emitter laser diodes (sources) and photodiodes (detectors) as well as microlenses and prisms required for the complete miniaturized sensor package (A. Pique et al.) LED Array by Laser Direct Write on Polyimide ~ 250 µm The bare die LEDs are 100 µm thick and the polyimide substrate is ~125 µm thick. Alberto Pique, NRL Integration of Microfluidic Valves Reviewed in Gervais et al. in Adv. Mater. 2011, 23, H151–H176 (Fig 6). Integration of Microfluidic Pumps From Gervais et al. in Adv. Mater. 2011, 23, H151–H176 (Fig. 7) Immunodiagnostics liked to GPS and telecommunicaions Explosive detection using unmanned underwater vehicle (Remus) •Displacement immunoassay on a chip •No reagent additions •Continuous monitoring for days •Assays directly in sea water •Optical readout transmitted to PDA •Coordinated with onboard GPS data Anne Kusterbeck, PI (NRL) V. Simplified Fabrication Technologies • • • • • Soft lithography CNC milling Laser ablation Laser direct write Hot embossing Materials Hard: – Glass – Silicon – Thermoplastics • Soft: – PDMS – Hydrogels – Teflon – Thermoplastics – Paper Surface modifcation: --antifouling --hydrophilic/hydrophobic --tethers for biomolecules --metal-coatings --textured VI. Miniaturization in electronics & communication • • • • • Personal communication Personal data access (e.g. IPhone, IPad) Cloud computing Agile networks On-chip energy sources VII. Systems Integration Choices Choices dictated by application and user. Choices are interconnected. Results must be accurate, timely & actionable. • • • • • • Integrated or off-chip optics/electronics Option of on-chip pumps and valves Automated sample processing Automated analysis Integrated data processing Telecommunications for data transfer Examples of System Choices • Automated detection using antibody array – MBio diagnostics for resource-limited settings – Leopard array immunosensor for food safety • Microflow cytometry – Analysis using coded beads • Multiplexed detection of biothreat agents • Clinical diagnostics or environmental monitoring – Continuous monitoring of marine algae • MBio Diagnostics, Inc., Boulder, CO • One sample, panel of results, in minutes – Multiplexed assays at point-of-care – Blood: HIV, Hepatitis, … – Respiratory: Flu, Strep, … – Cardiac: Troponin, … – Cell counting: CD4, … Disposable reagents, fluidics, waveguide Handheld reusable optics, electronics Inexpensive, battery operated © 2011 MBio Diagnostics, Inc. MBio Multiplexed Serology System © 2011 MBio Diagnostics, Inc. Antenatal Screening Panel HIV Hepatitis Syphilis Replace three rapid tests • >1 million deaths: congenital HIV & syphilis • Trial: Antenatal clinics in Kenya, 2700 patients © 2011 MBio Diagnostics, Inc. MBio Cell Counting System Brightfield Fluor1; CD3+ Fluor2; CD4+ ~500 um Three registered images Differential staining © 2011 MBio Diagnostics, Inc. 0.5 mm MBio CD4+ Cell Count Current Clinical Sample Data MBio CD4+ Count (cells/ul) 1500 1000 MBio Identity Passing Bablok 500 0 0 500 1000 1500 CD4+ Count (cells/ul) Flow Cytometry (BD FACSCalibur) © 2011 MBio Diagnostics, Inc. Contact: • Chris Myatt, CEO – firstname.lastname@example.org • Mike Lochhead, Vice President – email@example.com Opportunity: Food Safety Hanson Technologies’ OmniFresh 1000™ • Same automation as NRL Array Biosensor • Integrated with large-volume concentrator • Screens entire produce lots • Continuously samples produce wash • Results in 2 hours • Large samples (100-300 gallons) • Concentrates and then detects microorganisms using array biosensor • Two highly successful validation pilots completed on production washing 36 machines Microflow Cytometry based on Hydrodynamic Focusing • • • • Grooves direct sheath fluid above and below core stream Simulations and confocal images match closely Number of chevrons determines height of core Relative flow rates determines width of core Confocal Validation of Core Geometry Top view Side view Cross section Multiplexed Immunoassays Coded Beads E. Coli 73 Salmonella 77 58 Bead Array 32 Assays for detection of multiple targets Listeria Camphylobacter Different antibodies on each bead enables deeply multiplex detection Capture bead Bioagent Labeled antibody Color analysis 2 Fluorescent ID Tags Light Scatter Phycoerythrin Tracer Cholera toxin assay 0.7 6000 0.6 5000 0.5 0.4 3000 0.3 Luminex Microcytometer 4000 bead 81: CTX bead 75: BSA Luminex bead 81: CTX 2000 0.2 1000 0.1 0 0.0001 0 0.001 0.01 0.1 1 Toxin concentration (ng/ml) 10 100 1000 NRL 12-Plex Assay for E. coli O157:H7 10 96 96 92 92 71 71 75 75 100 100 79 79 77 77 81 81 1 50 50 54 54 56 56 58 58 Normalized PE >3 0.1 0.01 0.1 1 10 50 – g α Listeria 54 – chicken IgY (+ control) 56 – g α E. coli O157:H7 58 – g α Salmonella 71 – r α Ricin toxin 75 – BSA (- control) 77 – m α F. tularensis 79 – r α Y. pestis 81 – r α Cholera toxin 92 – m α SEB 96 – g α B. anthracis 100 – r α Shigella Detection Limits Bead Sets and Limits of Detection Detection Limits – 10% serum Bead ID Analyte Detection Limits-buffer 50 Listeria 1x105 (cells/ml) 1x106 (cells/ml) 54 Chicken IgY Positive Control Positive Control 56 E.Coli 1x104 (cells/ml) 1x104 (cells/ml) 58 Salmonella 1x105 (cells/ml) 71 Ricin 1x10-2 (ng/ml) Not determined 75 BSA Negative Control Negative Control 77 F. tularensis 1x 105 (cells/ml) 1x106 (cells/ml) 1x 105 (cells/ml) 79 Y. Pestis 1x 105 (cells/ml) Not determined 81 Cholera toxin 1x10-1 (ng/ml) Not determined 92 SEB 1x100 (ng/ml) Not determined 96 B. Anthracis 1x 104 (cells/ml) 1x 105 (cells/ml) 100 Shigella 1x 105 (cells/ml) 1x 105 (cells/ml) E. coli Assay E. coli -10% serum normalized 1.6 Normalized Fluorescence 1.2 0.8 0.4 0 1.00E-01 -0.4 1.00E+01 1.00E+03 Concentration (cells/ml) 1.00E+05 1.00E+07 Automated Sample Prep Sample + Beads Biotinylated Antibody Cocktail PE-Streptavidin Magnetic Trap Howell et al. Patent Publication 20110188339, and international application PCT/US11/22942 Verbarg et al. 2012 Lab on a Chip. Advance Article DOI: 10.1039/C2LC21189K Changing Magnetic Field in Microchannel Capture and Release Automated Processing for E.Coli Detection •Stronger signal in less time • Less reagent required •Preconcentration if desired •Sample volumes flexible Black bars: automated processing: 5 min + 3 min reagent exposures Gray bars: manual processing with 5 min + 3 min reagent exposures White bars: manual processing with 30 min + 30 min reagent exposu Microflow Cytometer with Automated Sample Processing •Can be used for: sequential samples (clinical, food, grab) periodic monitoring (air, water) •On chip reagents, mixing, analysis •Off chip magnets, optics, pumps, data processing GE + NRL: Integrate Sample Prep with Microflow Cytometer on Disposable Chip for POC Sample Preparation Module Reservoir 1 Reservoir Reservoir 2 4 Microcytometer Module Sheath Fluid Optical Detection Fluidic Control Magnetic Trap Port 3 5 Reservoir Reservoir Waste Outlet Sheath Fluid Sheath Fluid Sample Sheath Fluid Automated Microflow Cytometer System for Marine Algae: System considerations • • • • • Core Size – ideally the diameter of the smallest expected particle. – Larger core size gives more throughput but poor variance (Algae are submicron to hundreds of microns) Particle velocity – higher velocity provides greater throughput. – Increases internal backpressures requires more power-intensive pumps – Faster signal processing requires more power-intensive microcontroller (Bigger algae are very dilute) Detector Dynamic range – Wider range improves algae discrimination – Requires decreasing electronic noise provided by more expensive/complex electronic parts – Alternative is to overlap detectors, which requires more space and power (Scatter can be misleading due to inclusions in algae, colors critical) Resolution – the best resolution occurs with 16 or more data points per peak. – Faster sample processing (more power-intensive electronics) – More data throughput (complex electronic design) – More expensive detectors – Extra data storage space (Data must be stored on board) Sensitivity – increase minimum light level detection – Complex optical schemes (expensive coatings and parts, can require a lot of space) (Total system must fit in 8’inc h diamter can) Microflow Cytometer to Detect Changes Microflow Cytometer Field trials in fall 2011 •Reconfigured for corrosion resistance •Integrated in can for submersion •Pressure tested •Cage-deployed off Oregon coast •Measured 3 distinct algael populations •NO LEAKS Undergoing further miniaturization • 1 µ Synechococcus • 12-85 µ Nitzschia d. • 8-32 µThalassiosira Accuri C6 N. Hashemi, J.S. Erickson, J.P. Golden, F.S. Ligler, Biosensors and Bioelectronics (2011) Optical Biosensors • Current – Commercial biosensors available for point of use – Initial use by defense and diagnostics industries – Multiple commercial transitions • Near term – Expanding sensing capability/information density – Miniaturization of automated systems • Future – Small, inexpensive, easy-to-use sensors: for diagnostics, environmental monitoring, food & water safety – Biosensors for distributed operations: multiple targets, continuous monitoring, networked response Thank You NRLers Chris Taitt Lisa Shriver-Lake Joel Golden Peter Howell Jeff Erickson David Mott George Anderson Kim Sapsford (now FDA) Alberto Pique Carl Villarruel Post Docs Miriam Ngundi Nadia Kulagina Abel Thangawng Dan Ateya Jason Kim Jason Wojciechowski Mansoor Nasir Nastaran Hashemi Yasenka Memisevic Sponsors Defense Threat Reduction Agency ONR/NRL DARPA NIH Corporate Partners Precision Photonics Research International Hanson Technologies Constellation Technologies BioIdent Life Point GE Global Research Creatv Microtech AND YOU FOR LISTENING!
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