2. Outline Similar to Mass Spectroscopy Raman Spectroscopy background Experiments of interest Process and Key components Results Analysis Techniques New Technology and Applications Relevance by comparison to current techniques Discussion
10. Rotavirus Experiment (2010) Propagated in MA104 cells; G and P genotype confirmed by Hemi-Nested RT-PCR (type-specific primers) Substrate: OAD silver nanorods at 71° tilt(like before) Equipment: RenishawinViaconfocal Raman microscope system 785 nm near-infrared diode laser included Sample Size: 1.0 µL evaporated at RT Spectral Read Time: 3 – 10 second accumulations Data Analysis: Partial Least Squares Discriminant Analysis (PLS-DA) Four Criteria: (1) rotavirus-positive or –negative, (2) strain, (3) G genotype, (4) P genotype – capsidproteins Results: Reproducible (baseline correction by taking first derivative of spectra) Spectra indicate rotavirus-positive or –negative (PLS Toolbox software) Strain and Genotype predicted with specificity and sensitivity (>96%)
11. Reproducible Results (RV3) Take first derivative of sample spectra (A) for reproducible baseline correction (B)
13. Classification Model Efficiency Summary of the PLS-DA cross-validation results for classification according to three different models based on the strain, G genotype and P genotype.
14. Surface Enhancement 104 up to 1014 intensity increase in Raman Shift Substrates include Ag, Au, Cu Silver nanorods applied to glass 71° tilt off axis for optimal binding Glass covered in thin Ag layer Intensity increase from molecules in close proximity to metal surface Electromagnetic field induced by laser excitation of substrate Conduction electrons oscillate together
15. Spectroscopy vs. Fluorescence Fluorescence – Absorbs light to an excited state; Measurements based on resonance and return to equilibrium Raman effect Photon passes through; Scatter pattern determines reading intensity Incident photon can be any frequency for excitation unlike fluorescence; Can have interference (from substrate) Raman shift - initiated by vibration, rotation or low –frequency mode change Spectral peaks indicate wavelength and intensity of scattered light where the individual peaks show bonding vibration, polymer chain vibrations, lattice modes
17. Basic Process Laser creates rough surface plasmons (Ag or Au) Electric field increases/Raman shift increases proportionally – Substrate creates electric field Measured scatter signal is significantly enhancedby adsorbing molecules to the rough surface (up to 1014-15) Enhancement factor makes it sensitive enough to recognize single molecules with great specificity
19. Oligonucleotide targeting SERS has been used to identify DNA and RNA sequences in viruses, bacteria, etc. Uses combinations of Au or Ag nanoparticles and Raman active dyes (example: Cy3) bound to the nucleic acids Forms a silver coating on nucleic acid regions with bound dye Only able to compare to reference spectra for quick idea– still need sequencing for full identification and validation Full-Organism SERS more common for spectral analysis
20. Substrate makes all the difference Silver nanofibers have recently been used in virology SERS – single virus particle sensitivity, for viral subtyping and detecting viral mutations (gene insertion or deletion)
21. Examples of Fingerprints Compiling a Library of Spectra for quick diagnostic comparisons will speed medicine and vaccine production processes based on strains, mutants, etc.
22. Analysis techniques PLS DA regression – Spectral differences Sample matrix affects reading – media, cells, etc. Need algorithms to account for this variation when developing databases of spectra for reference/analysis PLS Toolbox Software to cross-validate spectra Use 90% of spectra to classify remaining 10% accurately Sensitivity – measure of false negatives Specificity - measure of false positives
23. Results Silver nanorods - extreme enhancement factors Rapid results Nondestructive SERS spectra can be used to rapidly differentiate between types of virus and strains of the same viruses (small changes) – Reference spectra comparison Worthwhile to develop a reference library of vibrational Raman spectra fingerprints for rapid and accurate virus identification in small amounts Consistency based on substrate – need uniformity
43. References 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 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 Thomas Huser. Introduction to Surface Enhanced Raman Spectroscopy. Feb 6, 2007 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 Sam Fahmy. Silver bullet: UGA researchers use laser, nanotechnology to rapidly detect viruses. Nov 15, 2006 http://www.cdc.gov/flu/professionals/diagnosis/rapidlab.htm
