Near Infrared Surface Enhanced Raman Spectroscopy Ceh 11 3 2010


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Near Infrared Surface Enhanced Raman Spectroscopy Ceh 11 3 2010

  1. 1. Near-infrared Surface Enhanced Raman Spectroscopy<br />Charles Hall<br />IRR-ATCC<br />November 3rd, 2010<br />
  2. 2. Outline<br />Similar to Mass Spectroscopy<br />Raman Spectroscopy background<br />Experiments of interest<br />Process and Key components<br />Results<br />Analysis Techniques<br />New Technology and Applications<br />Relevance by comparison to current techniques<br />Discussion<br />
  3. 3. Spectral Fingerprint<br />Similar to Mass Spectroscopy except more diverse in its diagnostic applications<br />
  4. 4. Raman Spectroscopy<br />Fingerprinting the universe<br />Advantages:<br />Fingerprint spectra (molecular signature)<br />Structural orientation/conformation<br />Intermolecular interactions<br />Laser-based spectroscopy<br />Small Sample size (0.5 – 1.0 µL) – Clinical use<br />Test low concentration samples (single molecule sensitivity)<br />Used in detecting explosives, nuclear waste, water pollution, etc. – Useful for police officers, medical staff, forensic scientists<br />
  5. 5. The Experiment (2006)<br />Viruses: RSV (5 strains/mutants), rhinovirus, adenovirus, HIV, Influenza (3 strains)<br />Equipment: near-IR confocal Raman microscope system<br /> fiber-optic interfaced 785 nm near-IR diode laser<br />Kaiser Optical Holospecf/1.8-NIR equipped with a LN2-cooled CCD camera<br />Sample Size: 0.5-1.0 µL <br /> intact virus<br />Time: - 1 hr adsorption <br /> (optional)<br /> - 30-50 sec spectral<br /> collection time<br />Data Analysis: N/A<br />
  6. 6. Experimental Diagram<br />
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  8. 8.
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  10. 10. Rotavirus Experiment (2010)<br />Propagated in MA104 cells; G and P genotype confirmed by Hemi-Nested RT-PCR (type-specific primers)<br />Substrate: OAD silver nanorods at 71° tilt(like before)<br />Equipment: RenishawinViaconfocal Raman microscope system<br />785 nm near-infrared diode laser included<br />Sample Size: 1.0 µL evaporated at RT<br />Spectral Read Time: 3 – 10 second accumulations<br />Data Analysis: Partial Least Squares Discriminant Analysis (PLS-DA)<br />Four Criteria: (1) rotavirus-positive or –negative, (2) strain, <br /> (3) G genotype, (4) P genotype – capsidproteins<br />Results: Reproducible (baseline correction by taking first derivative of spectra)<br /> Spectra indicate rotavirus-positive or –negative (PLS Toolbox software)<br /> Strain and Genotype predicted with specificity and sensitivity (>96%)<br />
  11. 11. Reproducible Results (RV3)<br />Take first derivative of sample spectra (A) for reproducible baseline correction (B)<br />
  12. 12. Baseline Correction (lose MA104)<br />
  13. 13. Classification Model Efficiency<br />Summary of the PLS-DA cross-validation results for classification according to three different models based on the strain, G genotype and P genotype.<br />
  14. 14. Surface Enhancement<br />104 up to 1014 intensity increase in Raman Shift<br />Substrates include Ag, Au, Cu<br />Silver nanorods applied to glass<br />71° tilt off axis for optimal binding<br />Glass covered in thin Ag layer<br />Intensity increase from molecules<br /> in close proximity to metal surface<br />Electromagnetic field induced <br /> by laser excitation of substrate<br />Conduction electrons oscillate together<br />
  15. 15. Spectroscopy vs. Fluorescence<br />Fluorescence – Absorbs light to an excited state; Measurements based on resonance and return to equilibrium<br />Raman effect <br />Photon passes through; Scatter pattern determines reading intensity<br />Incident photon can be any frequency for excitation unlike fluorescence; Can have interference (from substrate)<br />Raman shift - initiated by vibration, rotation or low –frequency mode change<br />Spectral peaks indicate wavelength and intensity of scattered light where the individual peaks show bonding vibration, polymer chain vibrations, lattice modes<br />
  16. 16. Raman shift - Measures scattered light (rare event)<br />Elastic (Rayleigh) vs. Inelastic (Stokes)<br />Raman Spectroscopy<br />
  17. 17. Basic Process<br />Laser creates rough surface plasmons<br /> (Ag or Au)<br />Electric field increases/Raman shift increases proportionally – Substrate creates electric field<br />Measured scatter signal is significantly enhancedby adsorbing molecules to the rough surface<br /> (up to 1014-15)<br />Enhancement factor makes it sensitive enough to recognize single molecules with great specificity <br />
  18. 18. Laser Light source<br />Near – infrared wavelength: ~785 nm<br />Biological sample friendly<br />
  19. 19. Oligonucleotide targeting SERS has been used to identify DNA and RNA sequences in viruses, bacteria, etc. <br />Uses combinations of Au or Ag nanoparticles and Raman active dyes (example: Cy3) bound to the nucleic acids <br />Forms a silver coating on nucleic acid regions with bound dye<br />Only able to compare to reference spectra for quick idea– still need sequencing for full identification and validation<br />Full-Organism SERS more common for spectral analysis<br />
  20. 20. Substrate makes all the difference<br />Silver nanofibers have recently been used in virology SERS – single virus particle sensitivity, for viral subtyping and detecting viral mutations (gene insertion or deletion)<br />
  21. 21. Examples of Fingerprints<br />Compiling a Library of Spectra for quick diagnostic comparisons will speed medicine and vaccine production processes based on strains, mutants, etc.<br />
  22. 22. Analysis techniques <br />PLS DA regression – Spectral differences<br />Sample matrix affects reading – media, cells, etc.<br />Need algorithms to account for this variation when developing databases of spectra for reference/analysis<br />PLS Toolbox Software to cross-validate spectra<br />Use 90% of spectra to classify remaining 10% accurately<br />Sensitivity – measure of false negatives<br />Specificity - measure of false positives<br />
  23. 23. Results<br />Silver nanorods - extreme enhancement factors<br />Rapid results<br />Nondestructive<br />SERS spectra can be used to rapidly differentiate between types of virus and strains of the same viruses (small changes) – Reference spectra comparison<br />Worthwhile to develop a reference library of vibrational Raman spectra fingerprints for rapid and accurate virus identification in small amounts<br />Consistency based on substrate – need uniformity<br />
  24. 24. Raman Scanners (Present)<br /><ul><li> Handheld
  25. 25. Library of spectra stored within
  26. 26. Gives diagnosis/identification
  27. 27. Lightweight
  28. 28. Sensitive
  29. 29. Ability to work at long range
  30. 30. Currently used for pollutants, narcotics and forensics
  31. 31. Proposed for blood testing alternative – check glucose, cholesterol, uric acid, etc.
  32. 32. Used in Cancer screening (by type)
  33. 33. $15,000 currently in price- projected to drop to $5,000
  34. 34. Recognize up to 10,000 chemicals</li></li></ul><li>Comparisons (CDC site)<br />Raman Spectroscopy<br />Alternatives (Flu)<br />Faster process<br />Tiny sample size required<br />Highly Sensitive/accurate<br />Each sample has a unique fingerprint or signature<br />Library of Spectra for diagnostics<br />Nondestructive to sample<br />Non-invasive for sampling<br />Wide range of conditions (temperature/pressure)<br />Structural information (spectra)<br />Potential to recognize mutations<br />Novel substrates (potential)<br />Direct Viral Culture – Time intensive (week)<br />Immunofluorescence (DFA or IFA) – 2-4 hrs<br />RT-PCR/Sequencing – 2-4 hours (up to week)<br />Serology – Multiple weeks<br />ELISA – 2+ hours<br /><ul><li> All require substantial sample size
  35. 35. Not accurate/sensitive consistently
  36. 36. Waste sample or invasive to retrieve sample
  37. 37. Specific conditions for each assay (required)
  38. 38. No way to account for mutations
  39. 39. Prone to contamination issues
  40. 40. No way to optimize (like substrate changes)
  41. 41. False positives/False negatives
  42. 42. Rapid Diagnostic Outsource kits – very fast (10-15 minutes for most)</li></li></ul><li>Discussion<br />Would this assay be of benefit to ATCC?<br />Clinical or production usefulness?<br />Could help identify co-infection problems in samples<br />Is the spectral library a good thing… for medicine, research or for vaccine production?<br />Implications – HIV testing, rapid pandemic vaccine anticipation, mutation modelling, security<br />
  43. 43. References<br />SaratchandraShanmukh, Les Jones, Jeremy Driskell, Yiping Zhao, Richard Dluhy, and, Ralph A. Tripp.Rapid and Sensitive Detection of Respiratory Virus Molecular Signatures Using a Silver Nanorod Array SERS SubstrateNano Letters20066 (11), 2630-2636 <br />Driskell JD, Zhu Y, Kirkwood CD, Zhao Y, Dluhy RA, et al. 2010 Rapid and Sensitive Detection of Rotavirus Molecular Signatures Using Surface Enhanced Raman Spectroscopy. PLoS ONE 5(4): e10222. doi:10.1371/journal.pone.0010222 <br />Thomas Huser. Introduction to Surface Enhanced Raman Spectroscopy. Feb 6, 2007<br />Jeremy D. Driskell, Yu Zhu, Carl D. Kirkwood, Yiping Zhao, Richard A. Dluhy, Ralph A. Tripp, Ron A. M. Fouchier. Rapid and Sensitive Detection of Rotavirus Molecular Signatures Using Surface Enhanced Raman Spectroscopy. PLoS ONE, 2010; 5 (4): e10222 DOI<br />Sam Fahmy. Silver bullet: UGA researchers use laser, nanotechnology to rapidly detect viruses. Nov 15, 2006<br /><br />