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

RAMAN SCATTERING Resonance Raman scattering, zesílení

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. (Northwestern University, 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

A metal – vacuum interface Surface plasmons (SP) are special electromagnetic surface waves which may be excited at a metal - dielectric interface. 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 b a 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 duration 7 ml of Ag colloid prepared by 15 min ablation time

ADVANTAGES OF SERS SPECTROSCOPY 1.Low sample concentrations Chemical analysis Study of structure and function of biomolecules Kall et. al. (1999) hemoglobinKneipp et. al. (1997) adenineNie et. al. (1997) rhodamine

ADVANTAGES OF SERS SPECTROSCOPY 2. Fluorescence quenching Raman 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 selectivity Raman 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 acidACTIVEINACTIVE Naphtalene ACTIVEINACTIVE Salicylic acid ACTIVEINACTIVE Nicotinic acid ACTIVEACTIVE Nicotinamide ACTIVEINACTIVE Adenine ACTIVEACTIVE Uracil ACTIVEACTIVE 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 adenine uracil

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

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

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., [ 3, 5, 3 (1999)

SERS SPECTRA FROM LIVING CELLS

Viets C, Hill W J RAMAN SPECTROSC 31: (7) JUL 2000 FIBRE-OPTIC SERS SENSORS 200  m Gessner et al. Biopolymers 67, 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 PORPHYRINMETALATED PORPHYRIN 5, 10, 15, 20-tetrakis(1-methyl-4-pyridyl) porphyrin (H 2 TMPyP) Ag +

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 H 2 TMPyP (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)

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 , 221 (1999) Metalation kinetics of H 2 TMPyP 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.

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

GOLD SURFACESSILVER SURFACES

5,10,15,20-tetrakis (1-methyl-4-pyridyl) porphyrin (TMPyP) GOOD SPECTRA FROM GOLD AND SILVER SERS spectra of 1 m M H 2 TMPyP obtained from silver (a) and gold (b) surface (Baseline corrected and Raman signal of glass subtracted ) Prochazka, M. et al. Biopolymers 2006, 82, 390 MacroRaman 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) MacroRaman nm

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

Integrovaný Ramanův systém s optickým mikroskopem HR 800