Chem-Bio Detection Approaches at PNNL Michael Lind Battelle

Chem-Bio Detection Approaches at PNNL Michael Lind Battelle, Pacific Northwest National Laboratory

Chem–Bio Detection Grand Challenges Homeland Security Driven Field portable, multiple sensor chem, bio detection platforms with onboard data analysis, data fusion and statistical interpretation Fully integrated, multiple agent detection from sampling to low false positive recognition in complex, highly cluttered environments Remote communication, fully automated, low maintenance, low expendables in marine and aerosol environments Fast, cheap, small, reliable 2

New Approaches for Collection, Separation and Detection Micro-beads for multifunction reactions and detection Molecular and bio imprinting for cells, proteins and nucleic acids Surface functionalized for bio-recognition using antibodies and enzymes Carbon nano-tube based concentrators and reactors using stabilized enzymes and selective sorbent nanomaterials Supercritical fluid extraction of biomarkers Miniaturized detection based on suspension array flow cytometry, biochip fluorescence arrays, mass spectroscopy 3

Chem-Bio Detection Context Sensing & Detection High-Volume Collection and Initial Separation Purification & Concentration Extraction & Separation Pre-Concentration Physical Environment Control Analysis & Data Fusion Communications Transduction & Visualization & Conditioning Interpretation & Knowledge 4

Core Research at PNNL A multi-disciplined approach Identification of Biomarkers Proteomics (Lipton/Wahl/Smith) Nucleic acids (Straub) Sample Preparation Molecular/Bio Recognition BEADS (Bruckner-Lea/Grate/Straub) Microfluidics (J. Wahl, Wunschel) Antibody production (Feldhaus, Siegel) Nucleic acids (Straub, Bruckner-Lea) Imprinted polymers (Harvey) Surface enzymes (Ackerman) Data analysis/integration Anderson/Jarman/Daly/Wunschel Transduction Methods Mass spectrometry, cantilever (Wahl, Grate) Fluorescence: Planar microarrays, bead suspension arrays (Straub, Bruckner-Lea) One-step transduction methods (Gutowska, Bruckner-Lea, Tarasevich) 5

Smart Materials are Key “Smart” materials for Compound-Specific l Collection Trapping and Analysis from Complex Sampling l Concentration Streams l Isolation/Separation l Detection l Measurement Functionalized surfaces Sampling Stream Flow l Three-Dimensional Molecular Smart Material Imprinting Target Analyte Detector Target Analyte Removal and Smart Material Regeneration l Stabilized Antibody Fragment Interactions Compound-Specific Trapping and Real- l Stabilized/Armored Enzymes l Functionalized Mesoporous Nanostructures Time Sensor Detection Smart Material Sensor 6

Hierarchically Constructed Sorbants Putting Nanoscience to Work Complexation Surface Monolayers Ordered Nanopores Macroscopic Support Sensors • High affinity ligands for target analytes • Nuclear • Chemical • Biological • ~3 binding sites/ nm 2 • Chemical selective • Shape selective • Optically responsive • High surface area ~1000 m 2/g • Nondendritic porosity • Rapid response • Particles (1 -200 mm) • Thin films • Monoliths 7

Multifunction BEADS for Biodetection Dirty Sample Large From Environment Volume Clean Detector Of Choice Tiny Volume Biochip Aerosol Liquid Cell Concentration Cell growth Or Induction Lysis Nucleic Acid Cleanup Flow Cytometer Nucleic Acid Amplification (optional) Protein Cleanup BEADS Mass Spec Micro. Cantilever Other 8

Beads: Moveable, Renewable Surfaces High surface to volume ratio for selective capture of cells or bio-molecules Interactive bead surface is delivered fresh for each sample l 10 -150 µm nonmagnetic particles l Polymer, hydrogel, glass, etc. , Spherical or non-spherical particles l 50 nm-10µm magnetic particles Compatible with biodetection and chemical sample processing ‘unit operations’ Operates within a fluidics architecture Example Antibody-based detection l 30 min-1 hour, automated l False positives and detection limit challenges as in all antibody assays. 9

Imprinted Media for Highly Selective Separation Molecularly imprinted polymers for highly selective capture from environment MIPs prepared by both block and suspension polymerization techniques Enormous matrix discrimination allows capture of relatively pure fractions that contain the target analyte enabling the development of compact field-portable instruments Affinity augmented bioimprinted magnetic beads selective capture of organism classes from the environment l Beads imprinted with Bacillus thuringiensis spores to capture and concentrate organisms of similar size and shape including anthrax spores l Material is capable of collecting genetically altered organisms and is compatible with direct analysis techniques such as MALDI MS Template Polymer After polymerization Extensive wash Molecularly imprinted polymer Lectin located in bioimprint site 10

Surface Functionalization for Bio Recognition via Immobilized Enzymes Highly concentrated active enzymes entrapped in functionalized mesoporous silica (FMS) are stabilized for at least 6 months (vs. few days in solution without FMS). Success so far with 3 different enzymes: organophosphorous hydrolase (OPH), glucose oxidase, glucose isomerase. Target applications include small-scale - robust, sensitive, lightweight sensors. J. Am. Chem. Soc. 2002, 124, 11242− 3 11

sc. Fv Antibodies & Yeast Display A Platform for Discovery and Evolution Step 1 Start with a library of 109 antibodies Step 2 Select out novel antibodies & evolve Step 3 Utilize for microarray detector at sub pg/ml Y Y Y Y Y Purify sc. Fv via flow cytometry Y Y Y Y Identify sc. Fv yeast clone with antigen specificity Y Y Y Y Y Y Botulinum toxin Before 3 n. M KD After 30 p. M KD 12 Y

Supercritical Fluid Extraction of Bacterial Spores and Analysis for Specific Biomarkers Speciation of bacterial endospores is difficult due to their nearly indestructible nature blocking unique biomarkers in the presence of background interference Supercritical fluids are a form of matter in which the liquid and gaseous states are nearly indistinguishable from one another Interface extraction process directly to selective absorbent for sample preparation and to detection process Objective is to miniaturize and field deploy utilizing MALDI TOFMS and Infrared spectroscopic techniques CO 2 + Modifiers, Me. OH, Formic Acid, Volatile Salts 13

Microcaltilevers A new high sensitivity transduction modality New research focused on multichannel surfaced functionalized microcantilevers Diving board structures operating the 10 KHz range. More sensitive than QCMs and SAW devices by several orders of magnitude now for gas phase detection. Extrapolated sensitivities for 100 MHz devices could extend to the 10 -21 gm level (single molecule) baring scaling problems Cantion 10 element array 14

Bacterial Identification by MALDI-MS Matrix-assisted Laser Desorption / Ionization Mass Spectrometry Provides broad spectrum analysis of bacteria (spores and vegetative), viruses, fungi, proteins based on specific proteins l Proven to species level, extend to strain level, based on cultural conditions l Fingerprint extraction works on mixtures Smaller 2” flight tubes built – make field portable MALDI-MS system feasible Sample #20: E. coli, P. agglomerans P. stutzeri. Mixture , 40 30 20 10 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 10000 Mass/Charge Ratio 15

Automated DNA Sample Preparation & Detection on Planar Microarrays Cell concentration using renewable separation column Detection cell lysis, DNA amp. Cell concentration onto microbeads DNA amplification (flow-through PCR) Automated system cleaning Microarray detection (offline) 10 cell/m. L detection limit in environment Low false alarm rates DNA identification 30 min to 3 hour sample to answer time Prototype E. coli 0157: H 7 strain identification (4 virulence markers), Campylobacter jejuni 16

Marine Biodetection Surface microlayer (top 50 microns) Research results have shown how the microlayer concentrates pollutants on the order of one million times greater than bulk water GPS drifters to measure physical parameters Floating buoy systems using micro array analysis E. coli O 157: H 7, Shigella, Campylobacter, Cryptosporidium, Giardia, Cyclospora, Microsporidium 17