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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. [email protected]
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.)