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 [email protected]ff.cuni.cz
Mar 21, 2016
Spektroskopie povrchem zesíleného Ramanova rozptylu a její využití při
studiu biomolekul
MAREK PROCHÁZKA
Divison of Biomolecular PhysicsInstitute of Physics, Charles University, Prague
CZECH [email protected]
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 eta l 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 SPECTROSCOPY1. 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 SPECTROSCOPY2. 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
year1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004
Num
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f 'SE
RS'
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ers
publ
ishe
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CI)
0
50
100
150
200
250
300
MO
SKO
VITS (R
EVIEW
)
Single molecular SER
S (KN
EIPP, NIE)
Analytical and biom
olecular applications
(CO
TTON
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RR
ELL)
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
+NCH3
N+ CH3
N+
CH3
+NCH3
+NCH3
N+ CH3
N+
CH3
AgN
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