Spektroskopie povrchem zes

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Spektroskopie povrchem zesíleného Ramanova
rozptylu a její využití pri studiu biomolekul
MAREK PROCHÁZKA Divison of Biomolecular
Physics Institute of Physics, Charles University,
Prague CZECH REPUBLIC prochaz_at_karlov.mff.cuni.cz
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RAMAN SCATTERING
Resonance Raman scattering, zesílení 103-105
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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.
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P a.E
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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
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ELECTROMAGNETIC versus CHEMICAL EFFECT
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SERS-ACTIVE SURFACES
Metal electrodes
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METAL COLLOIDS
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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
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ADVANTAGES OF SERS SPECTROSCOPY

• Low sample concentrations
• Chemical analysis
• Study of structure and function of biomolecules

Kall et. al. (1999) hemoglobin
Kneipp et. al. (1997) adenine
Nie et. al. (1997) rhodamine
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ADVANTAGES OF SERS SPECTROSCOPY
2. Fluorescence quenching Raman spectra of
fluorescent species, laser dyes, etc.
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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
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DISADVANTAGES OF SERS SPECTROSCOPY
1. Problem of active and inactive molecules
Compound b.-r. Ag colloid
c.-r. Ag colloid _________________________________
________________ Benzoic acid ACTIVE INACTIVE N
aphtalene ACTIVE INACTIVE Salicylic acid
ACTIVE INACTIVE Nicotinic acid
ACTIVE ACTIVE Nicotinamide ACTIVE INACTIVE Ad
enine ACTIVE ACTIVE Uracil ACTIVE ACTIVE
Wentrup-Byrne et al. Applied Spectrosc. 47, 1993,
1192
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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)
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DISADVANTAGES OF SERS SPECTROSCOPY
3. Interaction with metal surface changes
structural properties of adsorbed molecules
(photodecomposition, denaturation, etc.)
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Single molecular SERS (KNEIPP, NIE) ?
Analytical and biomolecular applications ?
(COTTON, GARRELL)
MOSKOVITS (REVIEW) ?
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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).
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SERS SPECTRA FROM LIVING CELLS
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FIBRE-OPTIC SERS SENSORS
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SINGLE MOLECULE DETECTION
Katrin Kneipp (Cambridge, USA)
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SINGLE MOLECULE DETECTION
Shuming Nie (Indiana University, USA)
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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.
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NANOSPHERE LITHOGRAFY USING DEPOSITE MASK
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GLASS-DEPOSITED COLLOID-ADSORBATE FILMS
B. Vlcková et al. (PrF UK)
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COLLOIDAL PARTICLES IMMOBILIZED ON
SILANE-MODIFIED GLASS SLIDES
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PORPHYRIN METALATION IN Ag COLLOIDAL SYSTEMS
5, 10, 15, 20-tetrakis(1-methyl-4-pyridyl)
porphyrin (H2TMPyP)
?
Ag
FREE BASE PORPHYRIN
METALATED PORPHYRIN
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SPECTRAL MARKERS OF PORPHYRIN METALATION
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PORPHYRIN METALATION (Quantitative analysis of
metalation process)
Hanzlíkova et al., J. Raman Spectr. 29, 575 (1998)
1. FACTOR ANALYSIS (singular value 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
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METALATION KINETICS (Influence of porphyrin
concentration and colloid properties)
Time dependent SERRS spectra of H2TMPyP (C1 mM
10 nM) 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
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METALATION KINETICS (as a probe of porphyrin
self-aggregates)
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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
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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, 351 base pairsporphyrin ratio)
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SERRS OF PORPHYRINS ON IMMOBILIZED METAL
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.
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GOLD SURFACES
SILVER SURFACES
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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 H2TMPyP obtained from silver
(a) and gold (b) surface (Baseline corrected
and Raman signal of glass subtracted)
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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)
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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
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Integrovaný Ramanuv systém s optickým mikroskopem
HR 800