Bimetallic Nanostars (Ag@Au) with High Surface Enhanced Raman Scattering (SERS) Performance: Detection of -Amyloid and Its Marker Thioflavin T

GARCÍA-LEIS, A.GARCÍA-RAMOS, J.V.

SÁNCHEZ-CORTÉS, S. jvicente.g.ramos@csic.es

INSTITUTO DE ESTRUCTURA DE LA MATERIA

SERRANO 121, 28006. MADRID-SPAIN

OutlineIntroduction

Fabrication and Characterization of SERS substratesNanostars

SynthesisCharacterization

Alzheimer Disease Markersβ-amyloid

Direct SERS DetectionDyes (Congo Red and Thioflavin T)

SERS characterizationThT - β-amyloid interaction

Indirect SERS Detection of β-amyloidTailoring the size and shape of Silver Nanostars

Ag@AuNS using AgNS as seeds

Conclusions

Surface Enhanced Raman Spectroscopy

0 R

Laser DetectorTwofold

Enhancement

Electromagnetic mechanism

Localized Surface Plasmon Resonance

Ag Au Cu

Metal nanostructures

Metal Colloids

--

-

-

---

--

--

-- ---

-- -

-

-

-----

Metal Electrodes Ag Ag+

Redox Cycles

Metal Films

Deposition: Evaporation, Laser photoreduction

Colloids

Need external aggregation to detect low concentrations

SERS sensitive substrates with complex morphology

Non reproducibility

Variation in signal/noise

Enhanced Electromagnetic Field

Nano-Stars and nano-spheres Fabrication

AgNO3 or HAuCl4* 3H2O

Hydroxylamine

CitrateBorohydrideHydrochloride

Hydroxylamine

NEW!!

!

Nano-StarsNS-Ag / NS-

Ag@Au

NanospheresA. Garcia-Leis, J.V. Garcia-Ramos and S. Sanchez-Cortes. JPC C. (2013) DOI: 10.1021/jp401737y

Characterization of nanoparticles

300 400 500 600 700 800 900

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

520

422

UV-vis.

/ nm

AgCT AuCT

Ext

intio

n

300 450 600 750 900 1050 12000,00

0,09

0,18

0,27

0,36

0,45

378

551

NS-Ag NS-Ag@Au

Ext

intio

n

/ nm

UV-vis.

50 nm 300 nm

AuCT(SEM)

AgCT(SEM)

Nanospheres

TEM

SEM

200 nm

TEM

SEM

Nano-stars(TEM and SEM characterization)

Nano-stars(TEM and SEM characterization)

SERS active substrates without aggregation!!

A. Garcia-Leis, J.V. Garcia-Ramos and S. Sanchez-Cortes. JPC C. (2013) DOI: 10.1021/jp401737y

[Ag+]/[HA]/[citrate]

A: 2.17/ 7.0 / 1.0

B: 0.21/ 7.0 / 1.0

C: 2.17/ 3.6 / 1.0

D: 2.17/ 7.0 / 0.0

Nano-stars(Plasmon characterization)

SERS active substrates without aggregation!!

A. Garcia-Leis, J.V. Garcia-Ramos and S. Sanchez-Cortes. JPC C. (2013) DOI: 10.1021/jp401737y

Nano-stars(Dark-Field scattering)

Nanospheres

Nano-stars

A. Garcia-Leis, J.V. Garcia-Ramos and S. Sanchez-Cortes. JPC C. (2013) DOI: 10.1021/jp401737y

Nano-starsSERS activity using probenecid (sulfamide) as probe molecule

SERS (λexc: 532 nm)

Raman in solid state (λexc= 1064 nm)

}

A. Garcia-Leis, J.V. Garcia-Ramos and S. Sanchez-Cortes. JPC C. (2013) DOI: 10.1021/jp401737y

Tailoring the size and shape of Silver Nanostars

Preparation of a colloidal suspension of Silver Nano Stars

Chemical reduction of Ag+ in two steps:

Step 1: Reduction agent is a neutral Hydroxilamine solution

Step 2: After a time T1, a 1% citrate solution is added. The great novelty of this method is the use of neutral HA and the no use of strong surfactant agents.

Experimental Conditions24 different samples of AgNS

[HA] 30.1 mM 60.2 mM 130 mM

[Ag+] 1.11 mM

V(CIT) 100 µL

T1 5 min.

60 m0.11 mM 10 µL

30 min. in.

[HA] = concentration of hydroxylamine solution [Ag+] = concentration of silver nitrate solution V(CIT) = added volume of 1% citrate solution T1 = the waiting time before adding the CIT solution

C)

B)A)

Extinction Spectra

Scheme of possible mechanism of growing nanoparticles based on TEM images.

Final morphology of Ag@AuNS using AgNS as seeds

532 nm (ThT resonant wavelength)

785 nm (ThT non-resonant wavelength)

Micrograph of Ag@Au NS obtained by TEM of A (A), B (B), C (C) and (D) is a general view of sample C. (E) SEM micrograph of Sample C. (F) EDX spectra of Sample C.

Extinction spectra (left) and TEM images (right) corresponding to the samples: A (a), B (b), C (c), D (d)

and E (e). Inset: Pictures of the colloids obtained by methods A, B, C,

D and E.

SERS spectra of ThT on Samples A and C exciting at 532nm (b and d, respectively) and 785nm on Sample A (a).

The extinction spectra of samples A (c) and C (e) and the absorption spectrum of ThT (0.1 M) in water (f) is also shown for comparison.

b) a)

c)

d) e)f)

Alzheimer disease

Congo Red Thioflavin T

Peptide with 36-43 aa

Histological dyes used to demonstrate the presence of amyloidal deposits in tissue

Neurodegenerative disease

β-amyloid

β–amyloid (1-42)

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala

1800 1600 1400 1200 1000 800 600

615

666

714

797

836

891

968

1003

1033

1060

1097

1129

119112

6113

0613

44

1400

1433

1483

1546

1597

117

328

438

551

622

690

801

849

924

979

1068

1172

1358

1433

1486

1522

1623

1694

1723

1804

1857

1907

2063

2114

2179

22892715

2763

278128

9129

26

2980

3069

3265

3328

365

528

692

72593

0

1070

113112

13

1329

1391

1460

1567

1605

1847

135

327

40649

855159

1

653

760

841

901

1003

1032

1123

1204

1264

1339

1445

1603

1669

2520

2545

2580

267427

3527

72

2931

3064

3166

323332

76

3432

1408

1413

1283

-Amyloid (1-42) Raman solid NSAg + -Amyloid (1-42) 2.22 M pH 9 NSAg + -Amyloid (1-42) 0.22 nM pH 9 NSAg + -Amyloid (1-42) 0.22 nM pH 7

exc.= 633 nm

660

1220

Inte

nsity

(a.

u.)

Wavenumber (cm-1)1058

SERS of β–amyloid(1-42) on Silver Nano-Stars

Wavenumber / cm-1

AssignmentsSolid Raman

(λexc.= 633 nm)

SERS NSAg pH 9 (633nm)

SERS AgCT pH 9 (785nm)

1669 vs - - Am I

- 1623 1623 (C=O)

1606 m - - Phe, (C=C)

1585 w 1595 1581 Phe, Tyr

1565 w 1558 1567 Am II

- 1483 1494 His

1469 sh 1463 - His (deprotonated)

1444 s 1433 1441 (CH2), (CH3)

1408 sh 1413 1401 (C-H)

- 1344 1347 tw(CH2) or (CH2)

1264 w 1261 1268Am III (β-sheet)

1236 w 1237 -

1185 vw 1191 - Phe, Tyr

1179 w 1172 1171 (C-C)

1123 w 1127 1128 v (C-C)

1091 vw 1091 - His, Lys, Arg

- 1068 1064 (C-C) aliphatic side chains

1031 m - 1034 Phe

1003 vs - 1001 Phe

969vw 968 961 op(=C-H)

1800 1600 1400 1200 1000 800 600

0

2000

4000

6000

130

225

511

688

728

927

961

1091

1329

1363

148116

021661

2874

31853203

3276

3306

3331

3386

3400

3423

1176

1397

1234

NSAg + -Amyloid 1-42 10-5mg/mL pH 9exc.= 532nm

621

1463

79810

60

1138

1190

1271

Inte

nsi

ty

Wavenumber (cm-1)

1555

Poor SERS spectra to be used for β-amyloid direct detection!!!

Use of Dyes to detect amyloid fibril formation

Congo Red

Thioflavin T

Nano-stars

1800 1600 1400 1200 1000 800 600

141

487

798944

1040

1156

1283

1375

1451

1593

NS-Ag AgHXCl

Inte

nsi

ty

Wavenumber / cm-1

SERS 10-6M

SERS 10-6M

FT-Raman 0,01M

Detection of Congo Red by SERS

NS-AgN

N

NH2

S

OO O

-

NN NH2

O O

SO

-

C=C

N=N

C-NC=C

NS-Ag

1800 1600 1400 1200 1000 800 600

143

532

67779

5

112813

94

1475

1543

2926

3417

NS@AgAu AuCT

FT-Raman ThT 0,01 M

SERS ThT 10-5M

Inte

nsi

ty

Wavenumber / cm-1

SERS ThT 10-6M

Detection of Thioflavin T by SERS

NS-Ag@Au

S

N+

CH3

NCH3

CH3

CH3

Cl-

NS-Ag@Au

CCCC=CCH3 C-N; CSC C-S

Detection by SERS

0,0 8,0x10-5 1,6x10-4 2,4x10-4 3,2x10-4 4,0x10-4

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

Is = I

sm*K

ad*[ThT]/(1+K

ad*[ThT])

R^2 = 0.97

Ism

1.43 ± 0.03

Kad

5,98E5 ± 0,87E5

Experimental data Theoretical adjustment

Ma

rke

r B

an

d In

ten

sity

[ThT] / mol*L-1

0,0 2,0x10-6 4,0x10-6 6,0x10-6 8,0x10-6 1,0x10-5

0

1

2

3

4

5

6

Is = I

sm*K

ad*[CR]/(1+K

ad*[CR])

R^2 = 0.98 Ism

6.42 ± 0.33

Kad

7,3E5 ± 1,2E5

Experimental data Theoretical adjustment

Ma

rke

r B

an

d In

ten

sity

[CR] / mol*L-1

CR ThT

Langmuir adsorption isotherm

0,0 2,0x10-64,0x10-66,0x10-68,0x10-61,0x10-5

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

Is = K

ad*I

sm*[ThT]

R^2 = 0.95 K

ad*I

sm= 1,2E5 ± 0,1E5M

ark

er

Ba

nd

Inte

nsi

ty

[ThT] / mol*L-1

0,0 2,0x10-84,0x10-86,0x10-88,0x10-81,0x10-7

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9Is = K

ad*I

sm*[CR]

R^2 = 0.98

Kad

*Ism

= 5,51E6 ± 0,05E6

SE

RS

Inte

nsity

[CR] / mol*L-1

Detection by SERS

SERS Sensitivity

Linear Region ][AnalyteIKI smadS

Congo Red Thioflavin T

600 800 1000 1200 1400 1600 1800

0

20000

40000

60000

80000

100000

120000

132

351

415 50

253

3

617

676

701

744 796

885

922

949

975

1007

1034

1068

1129

1155

1183

1212 12

58

1301

1338

1393

1444

1473

1521 1597

132

288

351

502

533

617

642

675

701 74

4

796

886

1007

1034

1068

1129

1155 11

92 1212

1261

1302

1327

1395

1413

1444 14

7515

0015

22

1598

10-4mg/mL

Inte

nsi

ty (a.u

.)

Wavenumber (cm-1)

NSAg + ThT (10-5M) + -Am (1-42)

10-5mg/mL

ThT- β-amyloid complex

The effect of [β-amyloid] on the SERS spectra on Ag nanostars

Conclusions A simple method of nanostructure fabrication with high

sensitivity in SERS technique has been developed.

This method allows higher reproducibility in SERS

measurements without aggregation.

Congo Red and Thioflavine T dyes were detected at

low concentrations.

The adsorption isotherm of ThT over nano-stars follows

a Langmuir model

SERS of -amyloid peptide has been obtained

through its interaction with ThT.

José V. Garcia-Ramos

M. VegaCañamares

Paz Sevilla

SagrarioMartínez-Ramirez

EduardoLópez Tobar

José A.Sanchez-Gil

Spectroscopy of Surfaces and Plasmon Photonics Group:

Adianez García-Leis

CSIC: Consejo Superior de Investigaciones Científicas

Instituto de Estructura de la Materia

Santiago Sánchez-Cortés

Elisa Corda Diego Romero Abujetas

Acknowledgements

Ministerio de Economía y Competitividad

Project FIS2010-15405

Grant: JAE-Pre 2011 (A.G-L.)