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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.

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spektroskopie povrchem zes len ho ramanova rozptylu a jej vyu it p i studiu biomolekul
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

raman scattering
RAMAN SCATTERING

Resonance Raman scattering, zesílení 103-105

surface enhanced raman scattering sers
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.

slide5

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

sers active surfaces
SERS-ACTIVE SURFACES

Metal electrodes

metal colloids

a

b

TEM, 80000x

METAL COLLOIDS
slide9

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

advantages of sers spectroscopy
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

advantages of sers spectroscopy1

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.

advantages of sers spectroscopy2
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

dis advantages of sers spectroscopy
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

dis advantages of sers spectroscopy1

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)

dis advantages of sers spectroscopy2

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.)

slide16

Single molecular SERS (KNEIPP, NIE) 

Analytical and biomolecular applications 

(COTTON, GARRELL)

MOSKOVITS (REVIEW) 

sers spectra from living cells

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).

slide19

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

single molecule detection
SINGLE MOLECULE DETECTION

Katrin Kneipp

(Cambridge, USA)

slide21

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)

slide23

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.

glass deposited colloid adsorbate films
GLASS-DEPOSITED COLLOID-ADSORBATE FILMS

B. Vlčková et al. (PřF UK)

porphyrin metalation in ag colloidal systems
PORPHYRIN METALATION IN Ag COLLOIDAL SYSTEMS

5,10,15,20-tetrakis(1-methyl-4-pyridyl) porphyrin (H2TMPyP)

Ag+

FREE BASE PORPHYRIN

METALATED PORPHYRIN

porphyrin metalation quantitative analysis of metalation process
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

metalation kinetics influence of porphyrin concentration and colloid properties
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

metalation kinetics as a probe of porphyrin nucleic acid complexes
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

metalation kinetics as a probe of porphyrin nucleic acid complexes1
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)

slide36

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.

slide37

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

slide38

GOLD SURFACES

SILVER SURFACES

slide39

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)

slide40

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)

slide42

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