1 / 44

Spektroskopie povrchem zesíleného Ramanova rozptylu a její využití při studiu biomolekul

Spektroskopie povrchem zesíleného Ramanova rozptylu a její využití při studiu biomolekul. MAREK PROCH Á ZKA Divison of Biomolecular Physics Institute of Physics , Charles University , Prague CZECH REPUBLIC p rochaz @karlov.mff.cuni.cz. RAMAN SCATTERING.

sven
Download Presentation

Spektroskopie povrchem zesíleného Ramanova rozptylu a její využití při studiu biomolekul

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Spektroskopie povrchem zesíleného Ramanova rozptylu a její využití při studiu biomolekul MAREK PROCHÁZKA Divison of Biomolecular Physics Institute of Physics, Charles University, Prague CZECH REPUBLIC prochaz@karlov.mff.cuni.cz

  2. RAMAN SCATTERING Resonance Raman scattering, zesílení 103-105

  3. SURFACE-ENHANCED RAMAN SCATTERING (SERS) P= a.E Fleischmann, M., Hendra, P.J. and McQuillan, A. J. (University of Southampton, UK) Chem. Phys. Lett. 1974, 26, 163. Albrecht, M.G. and Creighton, J.A. (University of Kent, UK, J. Am. Chem. Soc. 1977, 99, 5215 Jeanmaire, D.L. and Van Duyne, R.P. (Northwestern University, Evanston, USA) J. Electroanal. Chem. 1977, 84, 1 Moskovits, M. (University of Toronto, Canada) Rev. Mod. Phys. 1985, 57, 783.

  4. P= a.E

  5. ELECTROMAGNETIC EFFECT – SURFACE PLASMONS Surface plasmons (SP) are special electromagnetic surface waves which may be excited at a metal - dielectric interface. METAL Field pattern of a surface plasmon for two different wavelengths A metal – vacuum interface

  6. ELECTROMAGNETIC versus CHEMICAL EFFECT

  7. SERS-ACTIVE SURFACES Metal electrodes

  8. a b TEM, 80000x METAL COLLOIDS

  9. LASER ABLATION(preparation of “chemically pure” metal colloids) Prochazka et al., Anal. Chem. 69, 5103 (1997) Nd/YAG pulse laser, 1064 nm, 10 Hz repetition, 20 s pulse duration 7 ml of Ag colloid prepared by 15 min ablation time

  10. ADVANTAGES OF SERS SPECTROSCOPY • Low sample concentrations • Chemical analysis • Study of structure and function of biomolecules Nie et. al. (1997) rhodamine Kall et. al. (1999) hemoglobin Kneipp et. al. (1997) adenine

  11. Cotton et al. 1982, porphyrine Schwartzberg et al. J. Phys. Chem. B 2004, 108, 19191 ADVANTAGES OF SERS SPECTROSCOPY 2. Fluorescence quenching Raman spectra of fluorescent species, laser dyes, etc.

  12. ADVANTAGES OF SERS SPECTROSCOPY 3. Surface selectivity Raman spectra of adsorbed part of macromolecules Orientation of adsorbate molecules Fleischmann, M. et al. Chem. Phys. Lett. 1974, 26, 163

  13. DISADVANTAGES OF SERS SPECTROSCOPY 1. Problem of „active“ and „inactive“ molecules Compound b.-r. Ag colloid c.-r. Ag colloid _________________________________________________ Benzoic acid ACTIVE INACTIVE Naphtalene ACTIVE INACTIVE Salicylic acid ACTIVE INACTIVE Nicotinic acid ACTIVE ACTIVE Nicotinamide ACTIVE INACTIVE Adenine ACTIVE ACTIVE Uracil ACTIVE ACTIVE Wentrup-Byrne et al. Applied Spectrosc. 47, 1993, 1192

  14. adenine uracil 0 time (min)  500 DISADVANTAGES OF SERS SPECTROSCOPY 2. Problem of reproducibility of SERS spectral measurement Wentrup-Byrne et al. Applied Spectrosc. 47, 1993, 1192 (borohydride-reduced Ag colloid – right)

  15. carbon pyridine cyanide tyrosine tyrosine Otto A. J. Raman Spectrosc. 2002, 33, 593 DISADVANTAGES OF SERS SPECTROSCOPY 3. Interaction with metal surface changes structural propertiesof adsorbed molecules (photodecomposition, denaturation, etc.)

  16. Single molecular SERS (KNEIPP, NIE)  Analytical and biomolecular applications  (COTTON, GARRELL) MOSKOVITS (REVIEW) 

  17. Mitoxantrone (MXT) SERS SPECTRA FROM LIVING CELLS G.D. Sockalingum, S.Charonov, A. Beljebbar, H. Morjani, M. Manfait & I. Chourpa Int.J.Vibr.Spec., [www.ijvs.com] 3, 5, 3 (1999) After treatment of a cell population with the drug and incubation with colloids (step A), one cell is selected under the microscope and spectra are recorded at regular intervals along a line (step B). This line of spectra is shown in step C, where one axis represents the frequency domain (cm-1) and the other the points on the line. A different line is then recorded (either by a scanning laser or by moving the XY stage by 1-2 µm intervals).

  18. SERS SPECTRA FROM LIVING CELLS

  19. Viets C, Hill W J RAMAN SPECTROSC 31: (7) 625-631 JUL 2000 200 m m Gessner et al. Biopolymers 67, 2002, 327. FIBRE-OPTIC SERS SENSORS

  20. SINGLE MOLECULE DETECTION Katrin Kneipp (Cambridge, USA)

  21. AFM images of screened Ag nanoparticles. (A) Large area survey image showing four single nanoparticles.Particles 1 and 2 were highly efficient for Raman enhancement,but particles 3 and 4 (smaller in size) were not. (B) Close-upimage of a hot aggregate containing four linearly arranged particles.(C) Close-up image of a rod-shaped hot particle. (D)Close-up image of a faceted hot particle. SINGLE MOLECULE DETECTION Shuming Nie (Indiana University, USA)

  22. Time-elapsed video image of intermittent light emission recorded from a single silver nanoparticle. The elapsed time between images is 100 ms, and the signal intensities are indicated by gray scales.

  23. NANOSPHERE LITHOGRAFY USING DEPOSITE MASK

  24. GLASS-DEPOSITED COLLOID-ADSORBATE FILMS B. Vlčková et al. (PřF UK)

  25. COLLOIDAL PARTICLES IMMOBILIZED ON SILANE-MODIFIED GLASS SLIDES

  26. PORPHYRIN METALATION IN Ag COLLOIDAL SYSTEMS 5,10,15,20-tetrakis(1-methyl-4-pyridyl) porphyrin (H2TMPyP)  Ag+ FREE BASE PORPHYRIN METALATED PORPHYRIN

  27. SPECTRAL MARKERS OF PORPHYRIN METALATION

  28. PORPHYRIN METALATION (Quantitative analysis of metalation process) Hanzlíkova et al., J. Raman Spectr.29, 575 (1998) 1. FACTOR ANALYSIS (singularvalue decomposition algorithm) 2. Construction of SERRS spectra of PURE PORPHYRIN FORMS as a linear combination of subspectra 3. Determination of METALATION KINETICS as a time-dependent fraction of pure metalated porphyrin forms in the original spectra

  29. METALATION KINETICS (Influence of porphyrin concentration and colloid properties) Time dependent SERRS spectra of H2TMPyP (C=1mM – 10nM) adsorbed onto the three different Ag colloids  Metalation kinetics for each system and each C fitted by exponential function A) Metalation is limited only by the porphyrin concentration B), C) Metalation is limited mainly by porphyrin efficiency to remove residual ions from colloid surface

  30. METALATION KINETICS (as a probe of porphyrin self-aggregates)

  31. METALATION KINETICS (as a probe of porphyrin-nucleic acid complexes) Pasternack et al., Biochemistry, 22, 2406 (1983) UV-Vis absorption spectroscopy, CD etc. Poly(dA-dT) EXTERNAL BINDING Poly(dG-dC) INTERCALATION

  32. METALATION KINETICS (as a probe of porphyrin-nucleic acid complexes) Prochazka et al. J. Mol. Struct. 482-483, 221 (1999) Metalation kinetics of H2TMPyP and their complexes with nucleic acids adsorbed on laser-ablated colloid (0.5 mM porphyrin concentration, 35:1 base pairs:porphyrin ratio)

  33. SERRS OF PORPHYRINS ON IMMOBILIZEDMETAL COLLOIDAL NANOPARTICLES • solid surfaces (stability, reproducibility) • metal colloids (narrow and homogeneous particles size distribution) • metal nanoparticles immobilized on glass substrates Keating C. D. et al., J. Chem. Educ.1999,76, 949.

  34. APTMS MPTMS GOLD SURFACES 10% APTMS for 30 min 3-4 hours in citrate-reduced colloid (left to dry at 100 °C) SILVER SURFACES 20% APTMS or MPTMS for 30 min 6 hours in borohydride-reduced colloid

  35. GOLD SURFACES SILVER SURFACES

  36. 5,10,15,20-tetrakis (1-methyl-4-pyridyl) porphyrin (TMPyP) GOOD SPECTRA FROM GOLD AND SILVER MacroRaman 514.5 nm Prochazka, M. et al.Biopolymers 2006, 82, 390 SERS spectra of 1mM H2TMPyPobtained from silver (a) and gold (b) surface (Baseline corrected and Raman signal of glass subtracted)

  37. 5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrin (TSPP) GOOD SPECTRA FROM GOLD MacroRaman 514.5 nm Concentration dependence of SERS spectra of TSPP obtained from gold surface (Baseline corrected and Raman signal of glass subtracted)

  38. 5,10,15,20-tetraphenyl porphyrin (TPP) GOOD SPECTRA FROM SILVER MacroRaman 514.5 nm SERS spectra of 1mM TPP obtained from different spots of silver surface

  39. Integrovaný Ramanův systém s optickým mikroskopem HR 800

More Related