Pitt Conn 2012 Fi Cs As Invited Sers Talks Ba Assay


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Pitt Conn 2012 Fi Cs As Invited Sers Talks Ba Assay

  1. 1. Detection of single-digit Bacillus anthracis spores in ~12-min by SERS Frank E. Inscore, Atanu Sengupta, Chetan Shende Mike Donahue, Hermes Huang, Stuart Farquharson Funding NSF DARPA Materials and BSL-2/3 FacilitiesProf. Sperry (Chair Cell & Molecular Biology) Center for Biotechnology and Life Sciences University of Rhode Island Pittcon 2012www.rta.biz RTA Boothinscore@rta.biz Providing Chemical Information When & Where You Need It #2110
  2. 2. The Overall Need/ProblemDetection of Bioagents BA spores –almost perfect bioagent. Most likely to be employed by terrorist : • Intentional Dispersal by Terrorist right size so can be inhaled – most lethal route. • Domestic/Military Targets • e.g. weaponized aerosols, contamination of water & food supplies • B. anthracis, Y. pestis, F. tularensis, C. botulinum A, P. hantaDetection of Waterborne Pathogens • Unintentional Water Contamination • Domestic/Military • Cryptosporidium, Giardia, V. cholerae, Campylobacter jejuniDetection of Foodborne Pathogens • Unintentional Food Contamination • Domestic/Military • Listeria monocytogenes, E. coli (O157:H7), Salmonella entericaDetection of Clinical Pathogens • Unintentional Spread of Infections • Domestic/Military Hospitals • S. aureus (MERSA), Tuberculosis, AIDs RTA is developing complete package to address each of these application areas.
  3. 3. The Challenge & Goal Detect bioagents and pathogens on surfaces, in aerosols, water, in biofluids, and food. (Category A agents 1 st priority: Bacillus anthracis spores) The device must provide the following:• Sensitivity: Detect 10,000 spores Anthrax LD50 ~10,000 spores (100 ng)• Speed: Within 15 minutes• Specificity: Identify and discriminate pathogens (No False Positives!)• Reproducibility: Accurate and Repeatable (No False Negatives!)• Current methods: • DNA or RNA enumeration: (Culture growth - 24 hours) or Polymerase Chain Reactions (PCR, >4 hours) • Test kits (limited shelf life, very high false positive rate)
  4. 4. The Solution: SERSInherent specificity: all chemicals (drugs/metabolites) produce a uniqueRaman spectrum allowing unequivocal identification (no false-positives).Improved sensitivity: Ag and Au nanoparticle substrates used to generateSERS amplify Raman signals (increase scattering efficiency) by 1 million timesor more with potential detection at sub-ppb, i.e. 10-8 M (no false-negatives).100 mW, 1-min SERS 1ppm DPA (aq) SERS 1ppm DPA290 mW, 5-min RS 20,000 ppm DPA (aq) DPA (dipicolinic acid)100 mW, 5-min RS 83,000 ppm DPA (KOH) LMC = ~ 1 ppb RS290 mW, 5-min 83,000 ppm DPA RS solid DPA Enhancement Factor ~ 105 FT-R 785 nm
  5. 5. Surface-enhanced Raman Spectroscopy Raman, although weak effect, provides molecular specificity hν SERS BUT, when a molecule is within Ag provides a laser induced plasmon field, H N Single Molecule Detection H N N the efficiency of Raman scattering H N N H H some argue this requires increase’s by 10 6 i.e. 1 million times! Plasmon Field Surface-Enhanced enhancement factor (EF)Sub part-per million detection possible Raman Photon of 1012 -1014 ALSO, chemical contribution 3 Others say 105 -108 can provide additional 10 enhancement Sub part-per-billion detection becomes possible with SERS
  6. 6. Commercial SERS Substrates: Benzenethiol benzenethiol Conc. EF 10-3M 102 10-5M 104 10-8M 107 (~10ppb)RTA: LMC ~10-11M (0.01 ng/mL or 10 ppt)
  7. 7. Approach: RTA SERS Patented Sampling Systems provide instant response in seconds as opposed to 30-min or more! 2001: Simple SERS Sample Vials Molecules Sol-Gel Matrix Raman in Solution Scattering Laser Adsorbed silver gold Molecules Metal Particle2003: SERS Microplate 2004: SER-Active Capillary 2005: SERS spin-coated Disk 1 10High Throughput Screening Extraction and Pre-Concentration RTA Patents6,623,977; 6,943,031; 6,943,032; 7,312,088; 7,393,691; 7,393,692; 7,462,492; and 7,462,493
  8. 8. 2006: Functionalized Sol-Gel SERS (affords greater selectivity and sensitivity) PC OTC Std chromatographic media Primary chemically selective SER-active sol-gel (SG) sub-sets developed at RTA. Chem. Type Metal cap/ 800 µ Analyte Selectivity M Ag SG1 polar - negative silver SG2 non-polar - negative silver SG3 more non-pol - neg. silver SG6 very polar - neg. Ag Ag SG1-PDMS less polar - neg. silver SG2-PEG less non-polar - neg. silver SG2-PDMS more non-pol - neg. silver SG3-PDMS very non-pol - neg. silver SG4 very polar-positive AuPatents: 6623977, 6943031, 6943032, 7312088,7393691, 7393692, 7462492, 7462493, 7713914
  9. 9. R&D: SERS Lab-On-ChipDifferent wafer, glass and plastic LOC designs
  10. 10. RTA’s SERSID - Trace Chemical Analyzer’s for Field and Lab Use 2010 2011 Providing Chemical Information When & Where You Need It
  11. 11. Preliminary Analysis: Raman Spectroscopy B. cereus B. subtilis Core Wall Cortex (proteins-cysteine, B. anthracis (peptidoglycan) Ca dipicolinate) DNA 2- Ribosomes O O Ca 2+ O C N C O CaDPA Spore Coat ExosporiumPortable 1064 nm system: Chem ID, Hazmat & Hoax Material Analyzer Caveat: no consensus spectra in literature. Variability: growth/media conditions. Limitations: sensitivity/sample issues.J. Raman Spectrosc., 35, 82-86 (2004); Spectrosc., (2005)
  12. 12. Preliminary Analysis: Surface-Enhanced Raman Spectroscopy BC spores 0.03 mg/mL(aq) BS 0.01 mg/mL (aq) 80 mW 785nm 1-min 80 mW 785nm 1-min BS 0.01 mg/mL (aq) 160 mW 785nm 1-min Appl Spec, 58, 351 (2004) BC in DDA (78C) ~2-min BC in AA (22C) ~2-min * * BC in DDA (22C) ~60-min BC in 0.02M HNO3 (heat+sonicate) ~10-min BC spores in nasal mucus IJHSES, 20, 12-18 (2007) in AA BC spores in saliva in AA US Patent: 7713914 B2 SPIE, 5585, 53-57 (2005)
  13. 13. Microscope Image: Quantifying Spores in Counting Chamber Microscope Cell Counting: Counting Grid This Image = 60 spores Average for 10 grids = 87 spores Diluted by 10 Area is 0.2 mm x 0.2 mm Depth is 0.1 mm 87 spores/4 nl =2200 spores/microL Volume = 0.004 mm3 =21,725/microL = 4 nL 220 spores in actual volume measuredIJHSES, 20, 12-18 (2007)Bioterrorism, S. Morse, Ed., ISBN 978-953-307-636-2 (2012)
  14. 14. SERS: sensitivity great! 220 Spores detected ~2-min! 1 spore = 10 pg, DPA =10% spore weight 220 BC spores with AA on SG1PDMS 100 pg/microL (ppb) * DPA in AA reference spectrum Internal AA * Reference IJHSES, 20, 12-18 (2007)
  15. 15. Problem: need both high specificity & sensitivity 10^9 spores/mL + AA BC BS BAS RS SERS 1 mg/mL DPA (s) DPA (aq) Na2DPA (aq) Na2DPA (s) CaDPA (aq) CaDPA (s)
  16. 16. 16 The Proposal: SERS-Active Capture AssayIncorporate Molecular Recognition Elements for Specificity Pathogens Target Specific Molecular Recognition Proof of Concept: Elements Ag Nanoparticles NSF (July 2008) Sol-Gel Layer IJAC (March 2012) Glass Surface SPIE (March 2012) Dipicolinic acid 25 ppm B. cereus Patent Application: (2011) cysS-Ag 250 ppm B. subtilis No DPA spectrum same as before signature! adding BS
  17. 17. Peptide-Functionalized Silver Sol-gel • Ag nanoparticles immobilized within porous sol-gel in a glass capillary were successfully functionalized with a short peptide that specifically binds Bacillus anthracis spores. • Peptide functionalization and spore binding are verified by SERS. • The functionalized spectrum dominated by sulfur of the pep-cys-Ag bond (660 cm-1 ). tf = 0.5-72 hrsfunctionalize wash Use Immediately or Seal (stable >6-months) SERS of BA specific peptide pep-cys-Ag Peptide cys-Ag Cysteine-Ag Ag Sol-Gel Glass Capillary Wallpictures are not to scale
  18. 18. Assay: Spore Capture: Incubation and Wash A 5-step SERS assay was successfully developed. 1) Draw 10 µL sample into a peptide functionalized capillary, wait 5-15 minutes. 2) Perform washes to minimize non-specific interactions (40 sec) 3) Treat sol-gel capture matrix with new proprietary reagent wash (10-sec) 4) Perform additional treatment using AA wash (10-sec) incubation cleaning washes sol-gel & spore treatments Spore washes applied after spore incubation: minimize non-specific interactions within capture matrix while treatments amplify spore signal via DPA enhancement Peptide Ag Sol-Gel Glass Capillary Wall
  19. 19. Spore Detection & Sensitivity: SERS A 5-step SERS assay for BA was successfully developed. (also similar BC and BS assays) 5) Place capillary in Raman analyzer, measure spectrum (1 minute). Sensitivity: 10 spore spectrum! NO False Negatives! measuret = 60 seconds Total Assay: 7-17 min depends on spore incubation time (5-15 min) 1/100th of 1000 spores/mL sample is in capillary
  20. 20. Spore Assay SpecificityA 5-step SERS assay for BA was successfully developed. Selectivity: BA 100 spore assay challenged: 10-100X [higher] BC, BM, & BS. NO False Positives! BA-S BC BM BS 1/100th of 10000 BA spores/mL sample is in capillary
  21. 21. Spore Assay RepeatabilityA 5-step SERS assay for BA was successfully developed. Repeatability: 100 BA spores - 12 for 12 Capillaries! No False Negatives! 1/100th of 10000 spores/mL sample is in capillary
  22. 22. Reproducibility Assessed: ROC Curve Analysis 15-min incubation K=4.8 5-min incubation K=2.3 50/50 probability line Concentration Number of Mean 1007 Standard Mean Standard K Value Capillaries Peak Height Deviation Deviation (σ) Blank (BC,BM,BS) 9 0.02 0.18 0.1 104 BAS (5-min) 12 0.52 0.4 2.3 104 BAS (15-min) 3 0.96 0.12 4.8 Goal is a 95% confidence level (bioagent concentration that can be detected 95% of time). Determine K-value (indicates a statistically significant separation between a true and false response). K > 3.29 meet Army’s requirement (of 95% probability of detection & 5% probability of false alarm).Since some non-specificity of other Bacillus spores with sol-gel,use of BC blank as opposed to water blank is more realistic!
  23. 23. BA Assay Goal: IMPROVE PERFORMANCESpecific goal of this ROC analysis: determine if modifications reduce spore incubation time from 15 to 10 min, withoutcompromising sensitivity & specificity of the assay, i.e. detecting 10,000 BAS spores with 95% specificity (K value greater than 3.29). ACCOMPLISHED! K = 3.43 New Silver Sol-Gel Chemistry New Additional Post-Functionalization Wash New Spore Incubation Time 10-min @95% Confidence t = 10-min + 40-sec + 20-sec + 60-sec = 12-min Concentration Number of Mean 1007 Standard Mean Standard K Capillaries Peak Height Deviation Deviation (σ) Value Blank (105 BC) 7 0.02 0.0161 0.047 104 BAS (10-min) 7 0.181 0.078 3.43 ~12-min, Assay Specificity: 96% No False Positives/Negatives K = 4.19 Concentration Number of Capillaries Mean 1007 Peak Height Standard Deviation Mean Standard Deviation (σ) K Value Blank (Water) 5 0.025 0.0064 0.037 104 BAS (10-min) 7 0.181 0.078 4.19 ~12-min, Assay Specificity: 98.5% This clearly indicates that the non-specific interaction of BC with the BA assay substrate is around 2.5%. (∆=4%-1.5%)
  24. 24. Executive SummaryProposed device (SERS analyzer/BA assay) provides the following:• Sensitivity: Detect < 10,000 BA spores (as low as 10 BA-S spores)• Speed: Less than 15 minutes (within 12 minutes) Possible assay time = 8.5-min + 40-sec + 20-sec + 30-sec = 10-min!• Specificity: Identify and discriminate BA (against BC, BS, BM) (No False Positives!)• Reproducibility: Accurate and Repeatable (95% confidence 100 spores) (No False Negatives!) Complete publication for BA assay (ames and sterne) pending (2012). Assay validation at US Army Edgewood facilities (June 2012)