Quantum Dots as
Immunoassay Probes
Roger M. Leblanc
Introduction – quantum dots
CdSe QDs
Definition
(for most semiconductors)
PL Int., a.u.
• 1-10 nm length scale
(2.2, 2.6, 3.2, 4.3, and 5.5 nm)
Abs., a.u.
• A material that confines
electrons in 3D
400 λ, nm 700
400 λ, nm 700
Properties
CdSe QDs
• Size dependent emission
• Narrow emission band: fwhm 35 nm
• High quantum yields: ≤ 85 %
• Broad excitation spectra
• Chemical/photo stability
d =1.9 2.4 4.1 5.2 nm
λex = 365 nm
CdS-Peptide QDs – Cu2+ Detection
Peptide
HN
CH3
N
H3C
O
H2N
CH C
CH2 O
H
N
CH C
H
Cu2+ binding motif
O
H
N
CH C
CH
CH2 O
H
N
CH C
CH2
CH CH3
O
H
N
CH C
OH
CH2
Linker
CH3
Gly-His-Leu-Leu-Cys
SH
For synthesis
of QDs
Fmoc solid phase synthesis, MW = 541.67
H2O soluble, white powder, Yield = 79 %, Purity = 96 %
Gattás-Asfura, K. M. et al. Chem. Commun. 2003, 2684-2685.
CdS-Peptide QDs – Cu2+ Detection
Peptide
60 nm
S2Gly-His-Leu-Leu-Cys-CdS
24 Å
Aggregates visible in solution
TEM
521 nm
379 nm
400
λ, nm
PL Int., cps, a.u.
pH 7.5-8.5, [NaOH]
Abs., a.u.
[ 1x10-3 M ]
Cd2+
600
Gattás-Asfura, K. M. et al. Chem. Commun. 2003, 2684-2685.
CdS-Peptide QDs – Cu2+ Detection
Selectivity
+
4
Intensity, cps
2+
2+
2+
K , Mg , Ca , Co ,
2+
3+
Blank Ni , Fe ,
2+
2+
Zn , Cd
6.0x10
4
4.0x10
+
4
2.0x10
2+
+
Cu or Ag
0.0
450 500 550 600 650
λ, nm
Differentiation = AgCl, insoluble & undetected
Gattás-Asfura, K. M. et al. Chem. Commun. 2003, 2684-2685.
CdS-Peptide QDs – Cu2+ Detection
Selectivity, comparison
CdS-S-CH2-COOH
CdS-S-CH2-NH2
CdS-Cys
150%
150%
100%
100%
100%
50%
50%
50%
0%
0%
0%
150%
Ref.
K
Cu
Mg
Ca
Co
Ni
Fe
Zn
Cd
Ag
CdS-CLLGG
200%
150%
150%
150%
100%
100%
100%
50%
50%
50%
0%
0%
0%
Ref.
K
Cu
Mg
Ca
Co
Ni
Fe
Zn
Cd
Ag
200%
Ref.
K
Cu
Mg
Ca
Co
Ni
Fe
Zn
Cd
Ag
200%
X = Metallic ions
CdS-CLLHG
Ref.
K
Cu
Mg
Ca
Co
Ni
Fe
Zn
Cd
Ag
CdS-CGG
200%
Ref.
K
Cu
Mg
Ca
Co
Ni
Fe
Zn
Cd
Ag
Ref.
K
Cu
Mg
Ca
Co
Ni
Fe
Zn
Cd
Ag
200%
200%
Y = Relative PL intensity
CdS-Peptide QDs – Cu2+ Detection
Detected
Levels
at a Cu2+/QD molar
ratio of 17:1 (εQD =
2 x 104 M-1cm-1)
•
R2
= 0.99 exponential
fit (nM detection
capability)
4
4
PL Intensity, x10 cps
• Max. PL quenching
5
3
2
1
0
0
2
4
6
8
-6
[Metal Ions], x10 M
Gattás-Asfura, K. M. et al. Chem. Commun. 2003, 2684-2685.
10
Conclusions
• CdS-Cys-Leu-Leu-His-Gly QDs detected Cu2+ and
Ag+ with high selectivity and sensitivity
• Leu-Leu and His-Gly residues played a critical role
towards metallic ion selectivity
• Peptides facilitated fabrication of selective optical
sensor by way of amino acid sequence design
Gattás-Asfura, K. M. et al. Chem. Commun. 2003, 2684-2685.
CdSe QDs Composite Film – Paraoxon Detection
9 Layer-by-Layer technique (electrostatic interaction)
Quartz slide
Composite film
Chitosan
CdSe-S-CH2-COOH QDs
Organophosphorus hydrolase (OPH)
Constantine, C. A.; Gattas-Asfura, K. M.; Mello, S. V.; Crespo, G.; Rastogi, V.;
Cheng, T.-C.; DeFrank, J. J.; Leblanc, R. M. Langmuir; 2003; 19(23); 9863-9867.
CdSe QDs Composite Film – Paraoxon Detection
Film growth
0.06
0.05
0.04
0.03
0.02
0.03
0.01
0.00
4
Last
layer
(OPH)
7
6
5
4
3
200000
0.02
PL, Int., a.u.
6
5
8
0.04
Intensity, cps
Absorbance a.u.
Abs.,
a.u.
0.07
240000
0.05
7
Absorbance
0.08
0
1
2
3
4
5
6
7
8
Number of bilayers
3
2
1
0.01
160000
2
120000
1
80000
40000
0.00
400
400
500
600
λ, nm
λ, nm
1-7 = # QDs layers
700
700
0
540
540
560
580
600
620
λ, nm
λ,
nm
640
640
QD avg. size: 34 Å
Constantine, C. A.; Gattas-Asfura, K. M.; Mello, S. V.; Crespo, G.; Rastogi, V.;
Cheng, T.-C.; DeFrank, J. J.; Leblanc, R. M. Langmuir; 2003; 19(23); 9863-9867.
CdSe QDs Composite Film – Paraoxon Detection
Response of film upon exposure to paraoxon
0.12
OPH, H2O
OH
NO2
0.10
Abs., a.u.
O OH
P
+
O O
NO2
Absorbance, a.u.
O O
P
O O
0.08
7
6
5
0.06
4
3
0.04
0.02
2
1
0.00
Paraoxon, 1×10-7 M
250
250
300
350
λ, nm
λ, nm
400
450
450
1-7 (bottom-top) = 0, 0.5, 1, 2, 5, 10, and 15 min.
Constantine, C. A.; Gattas-Asfura, K. M.; Mello, S. V.; Crespo, G.; Rastogi, V.;
Cheng, T.-C.; DeFrank, J. J.; Leblanc, R. M. Langmuir; 2003; 19(23); 9863-9867.
CdSe QDs Composite Film – Paraoxon Detection
Response of film upon exposure to paraoxon
O OH
P
+
O O
NO2
OPH, H2O
OH
240000
200000
Intensity,
PL,
Int.,cpsa.u.
O O
P
O O
Blank
160000
a
10
min.
b
1×10-4 M
c
120000
NO2
1×10-9 M
d
e
80000
f
g
h
40000
0
540
540
560
580
600
λ,λ,nm
nm
620
640
640
CdSe QDs Composite Film – Paraoxon Detection
Epifluorescence images of film
895µm × 793 µm
Before exposure to
paraoxon
After exposure to
paraoxon
Conclusions
• QDs were successfully incorporated into composite
films via the layer-by-layer technique and
electrostatic interaction.
• The QDs/OPH ultra thin film selectively detected
paraoxon at the nM levels.
Detection of paraoxon using
QDs/OPH bioconjugate
Surface modification
-
O
-
P
P
O
ZnS
O
-
HS
CdSe
P O
O P
CH2 COO-
O
O
-
O
O
O
P
-
O
P
O O
S
O
ZnS
S
S
OO
OS
S
CdSe
S
S
S
O
O
S
O-
Hydrophobic
O
S
S
O
S
O
O
OO
O-
O O-
O-
+
P
O
Hydrophilic
Ji, X. J.; Zheng, J.; Xu, J.; Rastogi, V. K.; Cheng, T. C.; DeFrank, J. J.; Leblanc R. M. J. Phys. Chem. B 2005, 109, 3793-3799.
Detection of paraoxon using QDs/OPH bioconjugate
Bioconjugation
OPH
NH3+
-
O O
-
O
O
-
O O
-
O
S
O
ZnS
S
S
OO
S
CdSe
S
S
S
O
O-
S
O
OPH
O-
-
O
NH3+
O
NH3+-O
S
O
S
O
O
S
OPH
OO
-
O
S
O
O
pH = 7.3
NH3+
O
S
S
ZnS
S
O
S
NH3+ -O
S
+
H3N
OO
CdSe
NH3+
O O
S
O
ONH3+
O-
S
O-+
O H3N
O
O+H3N
S
S
O
+
H3N
O+H3N
S O
-
-
S
O
O+
NH3+H3N
OPH
(
OPH
NH3++H3N
O
O
S
OPH
+
H3N
O O+
H3N
OPH
= Active Site of OPH )
Isoelectric point of OPH : 7.6
Ji, X. J.; Zheng, J.; Xu, J.; Rastogi, V. K.; Cheng, T. C.; DeFrank, J. J.; Leblanc R. M. J. Phys. Chem. B 2005, 109, 3793-3799.
Detection of paraoxon using QDs/OPH bioconjugate
Secondary structure analysis of bioconjugates
OPH only
OPH/QDs bioconjugate
OPH/QDs bioconjugate in paraoxon
4
Molar Ellipticity Per Residue
2
-1
(deg*cm *dmol )
3x10
4
2x10
4
1x10
0
4
-1x10
4
-2x10
4
-3x10
190
200
210
220
230
240
250
260
Wavelength (nm)
CD spectra of pure OPH and OPH/QDs bioconjugates with and without
paraoxon. OPH concentration is 5 × 10-7 M in all cases. The OPH/QDs molar
ratio of bioconjugates is 10. Paraoxon concentration is 10-6 M
Ji, X. J.; Zheng, J.; Xu, J.; Rastogi, V. K.; Cheng, T. C.; DeFrank, J. J.; Leblanc R. M. J. Phys. Chem. B 2005, 109, 3793-3799.
Detection of paraoxon using QDs/OPH bioconjugate
Secondary structure analysis of bioconjugates
Percentage of Secondary Structure (%)
α-helix
β-strands
Turns
(T)
Unordered
(U)
αR
αD
βR
βD
OPH
40.03
25.53
2.40
4.43
13.67
15.53
OPH/QDs
bioconjugates
36.10
21.43
3.37
4.10
13.10
20.07
OPH/QDs
bioconjugates with
paraoxon (10-6 M)
30.33
30.30
17.33
7.90
22.33
8.13
Secondary structure data of OPH, OPH/QDs bioconjugates and
OPH/QDs bioconjugates in 10-6 M of paraoxon. The subscripts “R” and
“D” represent “ordered” and “disordered”, respectively.
Ji, X. J.; Zheng, J.; Xu, J.; Rastogi, V. K.; Cheng, T. C.; DeFrank, J. J.; Leblanc R. M. J. Phys. Chem. B 2005, 109, 3793-3799.
Detection of paraoxon using QDs/OPH bioconjugate
Fluorescence responses of bioassay
1.2
1.2
Normalized PL Intensity
1.0
0M
4.2 x
1.5 x
1.1 x
2.6 x
4.2 x
2.6 x
4.2 x
(a)
0.8
0.6
-8
10 M
-7
10 M
-6
10 M
-6
10 M
-6
10 M
-5
10 M
-5
10 M
1.0
Normalized PL Intensity
QDS:OPH=10:1
0.4
(b)
0.8
0.6
0.4
0.2
0.2
0.0
525
550
575
600
625
1.2
1.0
0M
-8
4.2 x 10 M
-7
1.5 x 10 M
-6
1.1 x 10 M
-6
2.6 x 10 M
-6
4.2 x 10 M
-5
2.6 x 10 M
-5
4.2 x 10 M
QDs only
(c)
0.8
0.0
525
550
575
600
625
Wavelength (nm)
Wavelength (nm)
Normalized PL Intensity
0M
-8
4.2 x 10 M
-7
1.5 x 10 M
-6
1.1 x 10 M
-6
2.6 x 10 M
-6
4.2 x 10 M
-5
2.6 x 10 M
-5
4.2 x 10 M
OPH:QDs = 100:1
Photoluminescence spectra of (a)
10:1 and (b) 100:1 molar ratio
OPH/QDs bioconjugates; (c) pure
QDs in different concentrations of
paraoxon. All samples were excited
at 350 nm.
0.6
0.4
0.2
0.0
525
550
575
600
625
Wavelength (nm)
Ji, X. J.; Zheng, J.; Xu, J.; Rastogi, V. K.; Cheng, T. C.; DeFrank, J. J.; Leblanc R. M. J. Phys. Chem. B 2005, 109, 3793-3799.
Detection of paraoxon using QDs/OPH bioconjugate
Fluorescence responses of bioassay
1.2
1.2
Normalized PL Intensity
1.0
0M
4.2 x
1.5 x
1.1 x
2.6 x
4.2 x
2.6 x
4.2 x
(a)
0.8
0.6
-8
10 M
-7
10 M
-6
10 M
-6
10 M
-6
10 M
-5
10 M
-5
10 M
1.0
Normalized PL Intensity
QDS:OPH=10:1
0.4
(b)
0.8
0.6
0.4
0.2
0.2
0.0
525
550
575
600
625
1.2
1.0
0M
-8
4.2 x 10 M
-7
1.5 x 10 M
-6
1.1 x 10 M
-6
2.6 x 10 M
-6
4.2 x 10 M
-5
2.6 x 10 M
-5
4.2 x 10 M
QDs only
(c)
0.8
0.6
0.4
0.2
0.0
525
550
575
Wavelength (nm)
0.0
525
550
575
600
625
Wavelength (nm)
Wavelength (nm)
Normalized PL Intensity
0M
-8
4.2 x 10 M
-7
1.5 x 10 M
-6
1.1 x 10 M
-6
2.6 x 10 M
-6
4.2 x 10 M
-5
2.6 x 10 M
-5
4.2 x 10 M
OPH:QDs = 100:1
600
625
Facts:
1. The quenching of the PL is
dependent to the concentration
of paraoxon
2. The quenching of the PL has a
maximum value in certain
range of concentrations
3. The decrease of the PL
intensity does not show a linear
relationship to the [paraoxon]
Ji, X. J.; Zheng, J.; Xu, J.; Rastogi, V. K.; Cheng, T. C.; DeFrank, J. J.; Leblanc R. M. J. Phys. Chem. B 2005, 109, 3793-3799.
Detection of paraoxon using QDs/OPH bioconjugate
Proposed mechanism of quenching
NO2
O
O P O
O
OPH
NO2
O
O P O
O
Secondary structure change
OPH
QDs
PL
Quenching
QDs
O
O P OH
O
+
HO
NO2
Detection of paraoxon using QDs/OPH bioconjugate
Effect of OPH:QDs molar ratio on the sensitivity of the bioassay
Quenching Percentage (%)
-6
Paraoxon, 4.2 x 10 M
-5
Paraoxon, 4.2 x 10 M
50
40
30
20
10
0
50
100
150
200
OPH:QDs (molar ratio)
Concentration effect of OPH to the sensitivity of bioassay
Ji, X. J.; Zheng, J.; Xu, J.; Rastogi, V. K.; Cheng, T. C.; DeFrank, J. J.; Leblanc R. M. J. Phys. Chem. B 2005, 109, 3793-3799.
Conclusions
• The OPH and CdSe(ZnS) QDs can form stable
bioconjugate through electrostatic interaction
• CD spectrum indicate a secondary structure change
of OPH in presence of paraoxon
• The intensity of photoluminescence of OPH/QDs
bioconjugate was quenched in presence of
paraoxon
• The secondary structure change of OPH was
responsible for the observed PL quenching
• Increasing the molar ratio of OPH over QDs will not
increase the sensitivity of OPH/QDs biosensor
Imaging the formation of β-sheet using QDs/peptide conjugates
HS
H
N
H 2N
O
N
H
O
H
N
O
N
H
O
H
N
O
N
OH
O H
N
S
O
N
H
O
O
SCH3
(A)
H
N
O
N
H
O
H
N
H
N
H 2N
O
N
H
O
H
N
N
SCH3
(A')
SCH3
O
O
N
H
O
OH
N
O H
N
S
O
N
H
O
O
H
N
H 2N
O
O
N
H
H
N
O
(C)
H
N
N
O
O
N
H
O
OH
O
(B')
(B)
HS
OH
O
SCH3
HS
O
O
N
H
SCH3
H
N
O
O
N
OH
O H
N
S
O
O
N
H
H
N
O
(C')
O
N
H
H
N
O
OH
O
SCH3
Structures of the synthetic peptides with cysteine linker and covalently bonded
Dansyl group: (A) NH2-Cys-Ile-Ile-Gly-Leu-Met-OH, (B) NH2-Cys-Ile-Ile-Pro-LeuMet-OH, (C) NH2-Cys-Leu-Ile-Met-Ile-Gly-OH, (A’) Dansyl-Ile-Ile-Gly-Leu-Met-OH,
(B’) Dansyl-Ile-Ile-Pro-Leu-Met-OH, and (C’) Dansyl-Cys-Leu-Ile-Met-Ile-Gly-OH
Imaging the formation of β-sheet using QDs/peptide conjugates
Schematic illustration of the surface coating of
synthetic peptide on TOPO-coated CdSe/ZnS
Imaging the formation of β-sheet using QDs/peptide conjugates
Steric configuration analysis by Circular Dichroism spectroscopy
1x10
NH2-Cys-Ile-Ile-Pro-Leu-Met-OH (Peptide B)
NH2-Cys-Leu-Ile-Met-Ile-Gly-OH (Peptide C)
0
5
-1x10
5
-2x10
5
-3x10
190
4
1.0x10
NH2-Cys-Ile-Ile-Gly-Leu-Met-OH (Peptide A)
200
210
220
230
240
Wavelength( nm)
(a)
250
260
Molar Ellipticity per Residue
2
-1
(deg*cm *dmol )
Molar Ellipticity per Residue
2
-1
(deg*cm *dmol )
5
Aβ(1-42)
Aβ(1-40)
3
5.0x10
0.0
3
-5.0x10
4
-1.0x10
190
200
210
220
230
240
250
260
Wavelength (nm)
(b)
Circular Dichroism spectra of (a) three model peptides and (b) full
length peptides.
Ji X., D,Naistat, C. Li, J. Orbulescu and R.M. Leblanc, Colloids and Surfaces B: Biointerfaces
2006, in press.
Imaging the formation of β-sheet using QDs/peptide conjugates
Secondary structural data of Aβ (1-40), and Aβ (1-42) peptides. The
subscripts “R” and “D” represent “ordered” and “disordered”, respectively.
Percentage of Secondary Structure
α-helix
β-strands
Turns Unorder
(T)
ed (U)
αR
αD
βR
βD
Aβ (1-40)
12.5
11.3
18.5
8.5
19.0
30.2
Aβ (1-42)
11.8
9.5
22.6
14.5
14.0
27.5
Ji X., D,Naistat, C. Li, J. Orbulescu and R.M. Leblanc, Colloids and Surfaces B: Biointerfaces
2006, in press.
Imaging the formation of β-sheet using QDs/peptide conjugates
Epifluorescence micrographs of 31-35 model peptides and controlled peptides
QDs-CIIGLM-OH
QDs-CIIPLM-OH
Dansyl-CIIGLM-OH
Dansyl-CIIPLM-OH
QDs-CLIMIG-OH
Dansyl-CLIMIG-OH
Ji X., D,Naistat, C. Li, J. Orbulescu and R.M. Leblanc, Colloids and Surfaces B: Biointerfaces
2006, in press.
Imaging the formation of β-sheet using QDs/peptide conjugates
Epifluorescence micrographs of full length peptides
Aβ (1-40) / QDs
mixture
Aβ (1-42) / QDs
mixture
Aβ (1-40) / Dansyl
acid mixture
Aβ (1-42) / Dansyl
acid mixture
Ji X., D,Naistat, C. Li, J. Orbulescu and R.M. Leblanc, Colloids and Surfaces B: Biointerfaces
2006, in press.
Conclusions
• The model 31-35 Aβ peptides with cystein linker
and Dansyl group were designed and synthesized
• A phase transfer approach was applied to
conjugate the peptides onto the surface of the QDs
• The steric configuration study by CD spectroscopy
revealed that the main secondary structural
component of the aggregation is β-strands
• Epifluorescence microscopic research showed that
QDs luminescent label approach showed a much
higher intensity and better contrast for imaging than
organic dye
PhD Graduate Students:
•
Jiayin Zheng (March 2006)
•
Kerim M. Gattás-Asfura (December 2005)
•
Xiaojun Ji (December 2005)
•
David Naistat (December 2005)
•
Changqing Li (June 2005)
•
Robert Triulzi
•
Jianmin Xu
•
Liang Zhao
Sr. Research Associate:
•
Jhony Orbulescu
Funding
National Science Foundation
U.S. Army Research Office