Spektroskopie povrchem zesíleného Ramanova rozptylu a její využití při

studiu biomolekul

MAREK PROCHÁZKA

Divison of Biomolecular PhysicsInstitute of Physics, Charles University, Prague

CZECH REPUBLICprochaz@karlov.mff.cuni.cz

RAMAN SCATTERING

Resonance Raman scattering, zesílení 103-105

Fleischmann, M., Hendra, P.J. and McQuillan, A. J. (University of Southampton, UK) Chem. Phys. Lett. 1974, 26, 163.

SURFACE-ENHANCED RAMAN SCATTERING (SERS)

Jeanmaire, D.L. and Van Duyne, R.P. (NorthwesternUniversity, Evanston, USA) J. Electroanal. Chem. 1977, 84, 1

Albrecht, M.G. and Creighton, J.A. (University of Kent, UK,J. Am. Chem. Soc. 1977, 99, 5215

Moskovits, M. (University of Toronto, Canada)Rev. Mod. Phys. 1985, 57, 783.

P = .E

P = .E

A metal – vacuum interface

Surface plasmons (SP) are special electromagnetic surface waves which may be excited at a metal - dielectric interface.

x

M etal D ie lectricm edium

1

Field pattern of a surface plasmon for two different wavelengths

METAL

ELECTROMAGNETIC EFFECT – SURFACE PLASMONS

ELECTROMAGNETIC versus CHEMICAL EFFECT

SERS-ACTIVE SURFACES

Metal electrodes

METAL COLLOIDS

ba TEM, 80000x

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 duration7 ml of Ag colloid prepared by 15 min ablation time

ADVANTAGES OF SERS SPECTROSCOPY

1. Low sample concentrationsChemical analysis

Study of structure and function of biomolecules

Kall et. al. (1999) hemoglobin Kneipp et. al. (1997) adenineNie et. al. (1997) rhodamine

ADVANTAGES OF SERS SPECTROSCOPY

2. Fluorescence quenchingRaman spectra of fluorescent species, laser dyes, etc.

Cotton et al. 1982, porphyrine

Schwartzberg et al. J. Phys. Chem. B 2004, 108, 19191

ADVANTAGES OF SERS SPECTROSCOPY

3. Surface selectivityRaman spectra of adsorbed part of macromolecules

Orientation of adsorbate molecules

Fleischmann, M. et al. Chem. Phys. Lett. 1974, 26, 163

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

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)

0 time (min) 500

adenineuracil

DISADVANTAGES OF SERS SPECTROSCOPY

3. Interaction with metal surface changes structural properties of adsorbed molecules (photodecomposition, denaturation, etc.)

Otto A. J. Raman Spectrosc. 2002, 33, 593

pyridine

cyanide

carbon

tyrosine

tyrosine

year

1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004

Nu

mb

er o

f 'S

ER

S' p

aper

s p

ub

lish

ed(S

CI)

0

50

100

150

200

250

300

MO

SK

OV

ITS

(RE

VIE

W)

Single m

olecular SE

RS

(KN

EIP

P, N

IE)

Analytical and biom

olecular applications

(CO

TT

ON

, GA

RR

EL

L)

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).

SERS SPECTRA FROM LIVING CELLS

Mitoxantrone (MXT)

G.D. Sockalingum, S.Charonov, A. Beljebbar, H. Morjani, M. Manfait & I. Chourpa Int.J.Vibr.Spec., [www.ijvs.com] 3, 5, 3 (1999)

SERS SPECTRA FROM LIVING CELLS

Viets C, Hill W J RAMAN SPECTROSC 31: (7) 625-631 JUL 2000

FIBRE-OPTIC SERS SENSORS

200 m

Gessner et al. Biopolymers67, 2002, 327.

Katrin Kneipp (Cambridge, USA)

SINGLE MOLECULE DETECTION

Shuming Nie(Indiana University, USA)

SINGLE MOLECULE DETECTION

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-up image 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.

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.

NANOSPHERE LITHOGRAFY USING DEPOSITE MASK

B. Vlčková et al. (PřF UK)

GLASS-DEPOSITED COLLOID-ADSORBATE FILMS

COLLOIDAL PARTICLES IMMOBILIZED ON SILANE-MODIFIED GLASS SLIDES

PORPHYRIN METALATION IN Ag COLLOIDAL SYSTEMS

FREE BASE PORPHYRIN METALATED PORPHYRIN

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

NH N

N HN

+NCH3

+N

CH3

N+

CH3

N+

CH3

+NCH3

+N

CH3

N+

CH3

N+

CH3

Ag

N

N N

NAg+

SPECTRAL MARKERS OF PORPHYRIN METALATION

PORPHYRIN METALATION (Quantitative analysis of metalation process)

3. Determination of METALATION KINETICS as a time-dependent

fraction of pure metalated porphyrin forms in the original spectra

2. Construction of SERRS spectra of PURE PORPHYRIN FORMS as a linear combination of subspectra

1. FACTOR ANALYSIS (singular value decomposition algorithm)

Hanzlíkova et al., J. Raman Spectr. 29, 575 (1998)

METALATION KINETICS (Influence of porphyrin concentration and colloid properties)

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

Time dependent SERRS spectra of H2TMPyP (C=1 M – 10 nM)

adsorbed onto the three different Ag colloids

Metalation kinetics for

each system and each C fitted by exponential function

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

R1

R3

R2

R4

NH N

N HN

Bx: Ry = +N

METALATION KINETICS (as a probe of porphyrin-nucleic acid complexes)

Poly(dA-dT)EXTERNAL BINDING

Poly(dG-dC)INTERCALATION

Pasternack et al., Biochemistry, 22, 2406 (1983)UV-Vis absorption spectroscopy, CD etc.

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 M porphyrin concentration, 35:1 base pairs:porphyrin ratio)

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.

APTMS MPTMS

SILVER SURFACES20% APTMS or MPTMS for 30 min

6 hours in borohydride-reduced colloid

GOLD SURFACES10% APTMS for 30 min

3-4 hours in citrate-reduced colloid(left to dry at 100 °C)

GOLD SURFACES SILVER SURFACES

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

GOOD SPECTRA FROM GOLD AND SILVER

SERS spectra of 1M H2TMPyP obtained from

silver (a) and gold (b) surface

(Baseline corrected and Raman signal of glass subtracted)

Prochazka, M. et al. Biopolymers 2006, 82, 390

MacroRaman514.5 nm

5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrin (TSPP)

GOOD SPECTRA FROM GOLD

Concentration dependence of SERS spectra of TSPP obtained from gold surface

(Baseline corrected and Raman signal of glass subtracted)

MacroRaman514.5 nm

5,10,15,20-tetraphenyl porphyrin (TPP)

GOOD SPECTRA FROM SILVER

SERS spectra of 1M TPP obtained from different spots of silver surface

MacroRaman514.5 nm

Integrovaný Ramanův systém s optickým mikroskopem HR 800