russian-italian seminar 11 december 2009 soft x-ray spectroscopy of bioorganic materials with 3 rd...

Russian-Italian seminar 11 December 2009

SOFT X-RAY SPECTROSCOPY OF BIOORGANIC MATERIALS WITH 3rd

GENERATION SYNCHROTRON SOURCES

Yan Zubavichus

Kurchatov Centre for Synchrotron Radiation and Nanotechnology

http://www.kcsr.kiae.ru/index.php


Biologically relevant materials:• Small molecules: amino acids, purine and pyrimidine

bases, simple carbohydrates, fatty acids, nucleoside phosphates, etc.

• Macromolecules: proteins, nucleic acids, polysacharides, lipids, etc.

• Supramolecular complexes: lipoproteins, ribosomes, etc.

• Organelles: membranes, nuclei, mitochondria, etc.

• Cells

• Tissues

• Biomimicking artificially designed nanostructured materials and devices (sensors, biochips, etc.)

Com

plexit

y


Soft X-ray synchrotron spectroscopy

(Photon Energy Range h < 1500 eV)Technique Information Possible modes/modifications Limitations/Drawbacks PES Photoemission Spectroscopy

Elemental composition, the number of chemically nonequivalent groups, film thicknesses, energy diagram of occupied electronic states in the VB

Core-levels/Valence band, angle-resolved, spin-resolved, resonant, photoelectron microscopy

Non-applicable to dielectric samples (powdered organics)

NEXAFS (XAS) Near-edge X-ray Absorption Fine Structure

“Fingerprints” of specific functional groups, energy diagram of unoccupied electronic states in the VB with element and orbital momentum specificity

Detection modes: transmission, total electron yield, partial electron yield, Auger electron yield, fluorescence yield, ion yield, etc.; linear/circular dichroism, edge-enhanced X-ray microscopy

XES X-ray Emission Spectroscopy

Energy diagram of occupied electronic states in the VB with element and orbital momentum specificity

Non-resonant / resonant (RIXS), linear/circular dichroism

For C,N,O K, the quantum yield is very low (core holes predominantly decay through Auger) long data acquisition times & exposure doses are required


Measurements: - BESSY II, Berlin, Germany (HE-SGM, PM1, UE52 PGM, U41 PGM)

- ALS, Berkeley, USA (8.0)


Severe experimental problem: Radiation damage

Soft X-ray induced decomposition of amino acids: an XPS, mass-spectrometry, and NEXAFS study, Y. Zubavichus, et al., Rad. Res. 161 (2004) 346.

395 400 405 410

N K-edge

9 min.

14 min.

pristine

Photon energy (eV)

4 min.

530 535 540 545

O K-edge

11 min.

17 min.

pristine

Photon energy (eV)

5 min.

284 285 286 287 288 289 290 291

C K-edge

30 min.

10 min.

20 min.

pristinea

Flu

ore

sce

nce

yie

ld (

a.u

.)

Photon energy (eV)

pristine

0 60 120 180

0.0

1.0x10-10

2.0x10-10

3.0x10-10

4.0x10-10

5.0x10-10

6.0x10-10

2 (H2)a

16 (NH2)

18 (H2O)

28 (N2, CO)

44 (CO2)

Exposure time (min.)

0 5 10 15 20 25 30 35 40 45 50 55 60

0.0

1.0x10-10

2.0x10-10

3.0x10-10

p=1.50x10-8 mbar

CO

CO2NH2

H2

Ion c

urr

ent, A

M/z

Gaseous decomposition products in the residual vacuum

Fast changes in PES & NEXAFS spectra


Experimental considerations to suppress or avoid damage

• Light source: BM is easier than undulator

• Fastest possible data collection

• Strict control of damage effects by successive scans

• New series – new spot on a sample (or continuous shift of the sample position during scan)

• Cooling or efficient heat sink


Combined PES + NEXAFS study: Amino acid Histidine (His) on Au(111)

Coordinatively active amino acid residue (often responsible for metal-binding in proteins)

Exists in a number of charge forms (mediator of electron/proton transfer in proteins)

Film 1 Film 2 Film 3 Evaparator temperature, ºC

105 115 115

Substrate temperature RT RT RT Deposition time, min 40 40 80 Film thickness, Å

ML 5.2 1

18.5 ~3

38.8 ~6

Surface chemistry of ultrathin films of histidine on gold as probed by high-resolution synchrotron XPSY. Zubavichus et al. JPCB 109 (2005) 884

H2N CH C

CH2

OH

O

N

NH

Preparation:Molecular Beam deposition with a Knudsen effusion cell


PES data

90 88 86 84 82

Abs

olut

e In

tens

ity [

coun

ts] Au 4f

534 532 530 528

O 1s

404 402 400 398

N 1s

290 288 286 284 282

C 1s

25 20 15 10 5 0

ValencebandFilm 1: Monolayer

Film 2: 3 Molecular layersFilm 3: 6 Molecular layers


280 290 300 310 320

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

90° 55° 20°

Inte

nsi

ty (

arb

. u

nit

s)

Photon energy (eV)

280 290 300 310 320

0.0

0.5

1.0

1.5

2.0

2.5

90° 55° 20°

Inte

nsi

ty (

arb

. u

nit

s)

Photon energy (eV)400 420

0.0

0.5

1.0

90° 55° 20°

Inte

nsi

ty (

arb

. u

nit

s)

Photon energy (eV)

390 400 410 420 430

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

90° 55° 20°

Inte

nsi

ty (

arb

. u

nit

s)Photon energy (eV)

520 530 540 550 560-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

90° 55° 20°

Inte

nsi

ty (

arb

. u

nit

s)

Photon energy (eV)

520 540 560

0.0

0.5

1.0

90° 55° 20°

Inte

nsi

ty (

arb

. u

nit

s)

Photon energy (eV)

Multilayer film

Linear Dichroism in NEXAFS

C K-edge

C K-edge

N K-edge

N K-edge

O K-edge

O K-edge

Monolayer film

No distinct polarization dependence no preferred molecular orientation


Au+ + + + + + + + + + +

Weak van der Waals interactions

- -

-

-

-+

Strong ionic-covalent bonding

Au

-

+

+

+

+

+

+

+

+ +

+

++

+

+

+

-

-

-

-

-

-

-

-

- -

-

-

- -

Multilayer film

Monolayer film

ZI His

His- anions


C,N,O NEXAFS library fornative amino acids

Innershell Absorption Spectroscopy of Amino Acids at All Relevant Absorption Edges, Y. Zubavichus, et al. J. Phys. Chem. A 109 (2005) 6998.

Sample preparation:“as is” powders pressed into

In foilNEXAFS data acquisition in the

partial electron yield mode


400 405 410 415 420 425 430

N K-edge

Photon Energy [eV]

285 290 295 300 305 310 315

285 286 287 288 289 290

Par

tial

Ele

ctro

n Y

ield

[a.

u.]

Photon Energy [eV]

C K-edge

530 535 540 545 550 555 560

O K-edge

Photon Energy [eV]

H2N CH C

CH2

OH

O

C

NH2

O

H2N CH C

CH2

OH

O

C

OH

O

H2N CH C

CH2

OH

O

H2N CH C

CH2

OH

O

OH

Aspartic acid (Asp) vs. Asparagine (Asn) Phenylalanine (Phe) vs. Tyrosine (Tyr)

Amino acids: NEXAFS fingerprints of specific functional groups


400 405 410 415 420 425 430

N K-edge

Photon Energy, eV

ZI-Gly (Gly1)

Gly+ Cl-

Na+ Gly-

NeutralGly (gas)

285 290 295 300 305 310

Gly+ Cl-

NeutralGly (gas)

Na+ Gly-

C K-edge

Par

tial

Ele

ctro

n Y

ield

, a.u

.

Photon Energy, eV

ZI-Gly (Gly1)

530 535 540 545 550 555

O K-edge

Photon Energy, eV

ZI-Gly (Gly1)

Gly+ Cl-

Na+ Gly-

NeutralGly (gas)

Effects of the charge state of an amino acid residue in NEXAFS

Solid-state NEXAFS spectra of glycine in various charge states, Y. Zubavichus, et al., J. Phys. Chem. B, 110 (2006) 3420

H2N CH C

R

O

O-

+H3N CH C

R

O

O-

+H3N CH C

R

OH

O

H2N CH C

R

OH

O

cation neutral molecule zwitter ion anion


Amino acids peptides: compositional effects

285 290 295 300 305

Phe

His

No

rma

lize

d A

bso

rba

nce

[a.u

.]

Photon Energy [eV]

His-Phe

280 290 300 310

Tyr

Photon Energy [eV]

LeuTyr LeuLeuTyr

Leu

280 290 300 310

Arg

Tyr

Gly

Photon Energy [eV]

GlyGlyTyrArg

The ‘building block” approach is valid to a certain extentNEXAFS spectroscopy of biological molecules: from amino acids to functional proteins, Y. Zubavichus, et al., NIMA 603 (2009), 111


290 300 310

287 288 289 290

FED

C

B DKP

Trigly

Digly

Gly

C1s

Inte

nsi

ty (

arb

. u

nit

s)

A

400 410 420 430

Photon Energy, eV

DKP

Trigly

Digly

Gly

N1s

530 535 540 545 550 555 560 565

DKP

Trigly

Digly

Gly

O1s

A

B

C D

NH2

CH

C

H

OH

O

NH2

CH

C

H

NH

O

CH

C

H

OH

O

NH2

CH

C

H

NH

O

CH

C

H

NH

O

CH

C

H

OH

O

H2C

NH

CH2

HN O

O

glycine diglycine tryglycine 2,5-diketopiperazine DKP(glycine anhydride – cyclic dipeptide)

Amino acids peptides: NEXAFS fingerprints of the peptide bonds

NEXAFS study of glycine and glycine-based oligopeptides, Y. Zubavichus, et al. J. Electr. Spectr. Relat. Phenom. 134 (2004) 25


400 410 420 430

*N-CH2

*N-CO

DKP FEFF8 model

DKP

Polytyr

Par

tial

Ele

ctro

n Y

ield

[a.

u.]

Photon Energy [eV]

N K-edge

Polyile

*N-CO

530 540 550 560

*O=C

O K-edge

*O=C DKP

FEFF8model

DKP

Polyhis

Polyile

One more step ahead: homopolypeptides (designed polymers composed of one sort of amino acids)

HN CH C

CH

HN

O

CH3

CH2

CH3

CH C

CH

HN

O

CH3

CH2

CH3

HN CH C

CH2

HN

O

OH

CH C

CH2

HN

O

OH

HN CH C

CH2

HN

O

N

NH

CH C

CH2

HN

O

N

NH

[ ] [ ] [ ]Polyisoleucine

PolytyrosinePolyhistidine

NEXAFS spectroscopy of homopolypeptides at all relevant absorption edges: polyisoleucine, polytyrosine, and polyhistidine, Y. Zubavichus, et al., J. Phys. Chem. B, (2007) 111 9803


Lysozyme (14.4 kDa)

Collagen (300 kDa)

BSA (66 kDa)

Ala

Arg

Asn

Asp

Cys

Gln

Glu

Gly

His Ile

Leu

Lys

Met

Phe Pro

Ser

Thr

Trp Tyr

Val

0

10

20

30

Fra

ctio

n, %

Lysozyme BSA Ovalbumin Collagen

285 290 295 300 305

ED

C

B

Lysozyme BSA Ovalbumin Collagen

C K-edge

A

400 405 410 415 420

E

D

C

B N K-edge

Inte

ns

ity

(a

rb. u

nit

s)

A

Towards functional proteins

Fractions of amino acid residues

Is X-ray absorption spectroscopy sensitive to the amino acid composition of functional proteins?, Y.Zubavichus, et al., J. Phys. Chem. B 112 (2008) 4478.

http://www.friedli.com/research/PhD/bsa/fig58c.gif


C,N,O NEXAFS library for nucleotide bases

N

NNH

N

NH2

NH

NNH

N

O

NH2

N

NH

NH2

O

NH

NH

O

O

NH

NH

O

O

Cytosine (C) Uracil (U) Thymine (T) Adenine (A) Guanine (G)

1

2

3

4

5

6

7

8

12

3

4

5

6

7

8

1

2

3

4

5

6

8

9

7 12

3

456

7

89

10

12

3

456

7

89

10

11

285 290 295 300 305 310 315

C

C K-edgeA

G

T

U

Nor

mal

ized

In

ten

sity

(ar

b. u

nit

s)

400 405 410 415 420 425

C

N K-edge

A

G

T

U

Photon Energy (eV)530 535 540 545 550 555 560

C

O K-edge

G

T

U

X-ray absorption spectroscopy of the nucleotide bases at the carbon, nitrogen, and oxygen K-edges, Y. Zubavichus, et al., J. Phys. Chem. B 112(44) (2008) 13711-13716.


Soft X-ray spectroscopic studies of solutions: 2 approaches

Microfluidic liquid cell with a thin window semi-

transparent to soft X-rays

Window material: Si3N4,

SiC (50-100 nm)

Microjet of a solution + efficient

differential pumping

Investigation of volatile liquid surfaces by synchrotron X-ray spectroscopy of liquid microjets, K.R. Wilson, et al., Rev. Sci. Instr. 75 (2004) 725

A liquid flow cell to study the electronic structure of liquidswith soft X-rays, O. Fuchs, et al., NIMA 585 (2008) 172

All techniques are available Only photon in – photon outtechniques (XES, FlY NEXAFS, RIXS)


Full O1s RIXS map of liquid water

Si3N4 Membrane(75 nm)

Isotope and Temperature Effects in Liquid Water Probed by X-Ray Absorption and Resonant X-Ray Emission Spectroscopy, O. Fuchs et al., PRL 100 (2008) 027801 & 249802

Two states : -Н-bonded intact water molecules -Ultrafast proton transfer induced by the oxygen core hole


Aqueous glycine: pH effects

N K-edgeNEXAFS

N KXES

SiC Membrane(75 nm)

Soft x-ray spectroscopyof organic molecules and liquids, O. FuchsPhD thesis (2009)

NH3+ NH2

NEXAFS XES


Conclusions• Synchrotron-based PES, NEXAFS, XES, and RIXS

are a highly efficient and informative set of experimental techniques to elucidate organic materials of biological significance in different forms, including bulk powders, thin evaporated films, adsorbates, aqueous solutions, etc.

• Radiation damage problems are serious but can be avoided

• The results presented above could be a good starting point for research projects with novel soft X-ray instrumentation at the KCSRNT


Acknowledgements

University of Heidelberg, Germany

University of Würzburg, Germany

University of Nevada, Las Vegas, USA

University of Graz, Austria

Prof. M.GrunzeDr. M.Zharnikov

Prof. E. UmbachDr. O. FuchsDr. L. Weinhardt

Prof. C. Heske

Prof. F. NetzerDr. G. Tzvetkov

russian-italian seminar 11 december 2009 soft x-ray spectroscopy of bioorganic materials with 3 rd...

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russianitalian seminar

ev slide

nanotechnology slide

complexity slide

nexafs c

amino acid histidine

tyrosine tyr amino acids

o nexafs library