Raman Spectroscopy and its Application in

Biology

Chandrabhas Narayana Chemistry and Physics of Materials

Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O., Bangalore 560064, India

cbhas@jncasr.ac.in http://www.jncasr.ac.in/cbhas

Lecture for TSU Seminar, February 19, 2013

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Light Scattering Laboratory

Study of

Physical

Properties of

Material

Raman

Spectroscopy

Brillouin

Spectroscopy

X-rays from

Synchrotron

Source

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Light Scattering Laboratory

• Elastic/acoustic properties of Nano systems (Nanotubes, Graphene) and bulk systems (pyrochlore, topological insulators, Li-Plastics Battery)

• Vibrational properties of Graphenes, Fast Ionic conductors, multiferroics, Metal Organic Framework solids, Molecular Solids etc.

• Ultra high pressure studies (both vibrational and structural) of ionic conductors, semiconductors, molecular solids such as Hydrogen, Silane, binary nitrides, Aluminium, multiferroics.

• Drug-Protein interactions, diagnostics using surface enhanced Raman spectroscopy

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What happens when light falls on a material?

Transmission

Reflection

Absorption

Luminescence

Elastic Scattering

Inelastic Scattering

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Why are Sky and Sea Blue? CASR

Why is this water not Blue? CASR

Why is some parts of the water in swimming pool Blue/not Blue?

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Vibrations in Molecules

HCl HF

H2O

NH3

SF6

Sym. Stretching

Asym. Stretching Sym. Bending

Asym. Bending

n1 = 3835 cm-1 n2 = 1648 cm-1 n3 = 3939 cm-1

n = 2991 cm-1 n = 4139 cm-1

n1 = 3505.7 cm-1 n2 = 1022 cm-1 n3 = 3573.1 cm-1

n1 = 774.55 cm-1 n4 = 523.56 cm-1 n3 = 947.98 cm-1

n5 = 643.35 cm-1 n6 = 348.08 cm-1 n2 = 615.02 cm-1

n4 = 1689.7 cm-1

8086 cm-1 = 1 eV

Room Temp = 25 meV

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Classical Picture of Raman

Stokes Raman Anti-Stokes Raman

Induced Polarization Polarizability

Taylor Series Expansion

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Energy diagram and

Quantum picture

Vibrational states Electronic states

Virtual states

g

ex

photon

<eg,p2|Her|p2,eb> <eb,p2|Hep|p1,ea> <ea,p1|Her|p1,eg>

|Es-Eb|x|Ei-Ea| S a,b

Raman cross section

If Ei = Ea or Es = Eb

We have Resonance Raman effect

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Raman spectra of CCl4

Sym. Stretching

asym. Stretching asym. Bending

Sym. Bending

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Raman spectra of CCl4

Isotope effect Cl has two isotopes 35Cl and 37Cl Relative abundance is 3:1

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RAMAN OF ISO-STRUCTURAL CRYSTALS

Diamond spectra is similar to the crystalline Silicon and Germanium but is shifted to higher Raman frequency because of lighter weight and bond strength.

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Raman, Fluorescence and IR

Scattering Absorption

and emission Absorption

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Problems with Raman:

a)Very Weak – for every 109 photons only 1

photon Raman

a)Resonant Raman not feasible with every sample.

b)Absorption a better process than scattering

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How do we see color? CASR

How do we see color? CASR

How do we see color?

Additive Substractive

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Plasmons in Nanoparticles CASR

Origin of

Surface Enhanced Raman Spectroscopy

• Initially – thought to be depended on Surface

Area of the rough surface

• It was shown that the scattering cross section far

exceeded the number of molecules on the rough

surface.

• It was proposed that the origin was due to

surface plasmons – hence a truly nano

phenomenon.

• Alkali and Ag the best, Au and Cu the next best,

Al, In, Pt followed by transition metals and then

bad conductors.

• Exciting wavelength, polarization, exact nature of

the nanostructure also effect the SERS

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Earlier SERS was thought to be due to creation of larger surface area

Now we understand it to orignate from the surface plasmon of the nano structures

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Surface Enhanced Raman Scattering

(SERS) Raman signal intensity gets enhanced when molecules are

adsorbed on metal nanoparticles, colloids, island films etc.

Pathways for enhancement :

Electromagnetic enhancement

Enhanced local optical fields of metallic nanostructure

Chemical enhancement

Molecule-nanostructure system provide new energy states

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Raman Spectrometers

Micro–Raman setup

US Patent No. US 8,179,525 B2 (2012), G.V. Pavan Kumar et al Current Science (2007) 93, 778.

Stage

Objective lens

Dichroic Mirror

Camera Edge filter

Focusing lens

Computer

Mono- chromator

CCD

Optical fiber

LASER

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New Nanoparticle Architecture for SERS application

Gayathri Kumari and Pavan Kumar G.V.

Gayatri Kumari and C. Narayana J. Phys. Chem. Letters 3, 1130 (2012)

G.V. Pavan Kumar et al. J. Phys. Chem. C 111, 4388 (2007)

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Gayathri Kumari

28

Ag Core – Au Shell

Nanoparticles with hot spots

100 nm 100 nm

100 nm

a) c)

b) d)

Au : Ag = 0.1 Au : Ag = 0.8

Au : Ag = 0.4

100 nm 100 nm

100 nm

a) c)

b) d)

100 nm 100 nm

100 nm

100 nm 100 nm100 nm100 nm 100 nm100 nm

100 nm

a) c)

b) d)

Au : Ag = 0.1 Au : Ag = 0.8

Au : Ag = 0.4

G.V. Pavan Kumar et al. J. Phys. Chem. C 111, 4388 (2007)

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29

SERS from Hot Spots

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1

0

5000

10000

15000

20000

25000

30000

a) Ad

Tp

Imd

ATP

Hb

Ram

an

In

ten

sit

y

Au : Ag

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1

0

100000

200000

300000

400000

500000

b)

Ra

ma

n I

nte

ns

ity

Au : Ag

Ad

ATP

TP

Imd

Hb

Ad – Adenine, TP – Thiophenol, Imd – Imidazole,

ATP – adenosine Triphosphate, Hb - Hemoglobin

G.V. Pavan Kumar et al. J. Phys. Chem. C 111, 4388 (2007)

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Gold Silica Silver Sandwich Structure

• The Silica layer between Gold and Silver can be the

buffer layer.

• The SERS enhancement was possible by controlling

the gold layer.

Triple layer nanostructure:

Shoute, L. C. T. et al Applied Spectroscopy, 2009, 63, 133

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Ag Ag

SiO2 Au

(a) (b) (c) (d)

Gold Silica Silver Sandwich Nanoparticles

Gayatri Kumari and C. Narayana J. Phys. Chem. Letters, 3, 1130 (2012)

TEM images at the different stages of formation of sandwich nanoparticles

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Gold Silica Silver Sandwich Nanoparticles

FESEM picture of Ag@SiO2@Au

Gayatri Kumari and C. Narayana J. Phys. Chem. Letters, 3, 1130 (2012)

SERS from Au-SiO2-Ag nanostructures

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SERS from Au-SiO2-Ag nanostructures

Gayatri Kumari and C. Narayana J. Phys. Chem. Letters, 3, 1130 (2012)

10-5 M of Thiophenol 10-7 M of Thiophenol

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Ag@SiO2@Au

SiO2@Au

Small Molecule Interaction with

Oncogenic Kinases, Aurora A and

Aurora B

Soumik Sidhanth, Partha P. Kundu, D. Karthikeyan, Tapas K. Kundu

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Soumik Sidhanth

Tapas Kumar Kundu

Soumik Siddhanta et al, RSC Advances (2013) in press

D. Karthigeyan et al, PNAS (USA) under Review

Dhanasekaran Karthigeyan

The relative localization of Aurora A and Aurora B in mitotic cells is shown above. The kinases play a role in cytokinesis too, where they are concentrated in the mid-body region.

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Overexpression of Aurora A by gene amplification or other means, leads to aneuploidy, which in turn leads to oncogenesis. Mitotic progression is disrupted and normal spindle orientation of chromosomes is not achieved. The cell fails to undergo cytokinesis, producing tetraploid progeny.

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Structure of Aurora A Kinase

N-Terminal

C-Terminal

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J. M. Elkins et al, Journal of Medicinal Chemistry 55, 7841-7848 (2012)

Structure of Aurora A Kinase

J. Nowakowski et al, Structure 10, 1659-1667 (2002)

Soumik Siddhanta et al, RSC Advances (2013) in press

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Structure of Aurora A Kinase

Aurora A and Aurora B are very much identical and have only 4 residues different in the catalytic part This makes it difficult to make specific Inhibitors for these molecules for these

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SERS spectra of Aurora A kinase and Aurora B kinase in presence of silver nanoparticles. λ= 632.8 nm; signal accumulation time = 30 s

Aurora A kinase Aurora B kinase

SERS of Aurora Kinase

Tyrosine Phenylanaline Amide I

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Soumik Siddhanta et al, RSC Advances (2013) in press

SERS spectra of Aurora A/Aurora B kinase (black) and denatured Aurora A/Aurora B kinase (red).

Effect of Denaturing of the Protein

Aurora A kinase Aurora B kinase

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Soumik Siddhanta et al, RSC Advances (2013) in press

Active sites of Aurora A kinase

This Aurora A-adenosine complex shows the hinge region (green), Glycine rich loop(red) and activation loop (purple).

The position of a conserved Threonine residue, Thr-288, which is phosphorylated during activation of Aurora A, is shown by a star.

Type I inhibitors bind to the ATP binding site through the formation of 1–3 hydrogen bonds to the kinase ‘‘hinge’’ residues and through hydrophobic interactions in and around the region occupied by the adenine ring of ATP.

Type II inhibitors use the ATP-binding site, and also exploit unique hydrogen bonding and hydrophobic interactions made possible by the DFG residues of the activation loop being folded away from the conformation required for phosphate group transfer

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Conclusions

• SERS can be used to validate the autodock and molecular dynamics simulations with the knowledge of the structure of the protein.

• SERS gives the interactions in the proteins in their active state, in physiological conditions.

• Thus helps in the derivatization of the small molecule for producing effective drug as well as fast screening of the potential molecules.

• With better control over molecular dynamics simulation, it opens up new avenues in the drug discoveries.

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Acknowledgements R. Dhanya Venkat Srinu Badram Soumik Sidhanth Gayathri K Partha Pratim Kundu Dr. Diptikanta Swain Dr. Gopal K Pradhan Dr. Pavan Kumar Dr. Kavitha G Dr. Md. Seikh Dr. Murugavel Dr. Navneeth Ms. Sonia Balan Dr. Nashiour Rohman Dr. Veena HG

POCE Students: Rangarajan Gayathri Nair Sruthi Vibha

Collaborators Tapas Kundu Ranga Uday V. Nagaraja Hans Agren

Funding sources: DST, DBT, JNCASR, Swedish Research Links