Quantum Dot Conjugatesfor Imaging Applications
Sungjee KimDept. of Chemistry
POSTECH
SlideQD as Bright & Tunable IR Emitter 1
(1) http://www.komabiotech.com.(2) Angew. Chem. Int. Ed. 2005, 44, 2508.
Quantum dot emission spectra: unpublished data
Lanthanide complexes(2)
- Limited emission wavelength tunability- Small absorption cross-section
Organic dye molecules(1)
- Low quantum yield at NIR & IR range because of molecular vibration modes
Quantum Dots at NIR & IR
Bright and wavelength-tunable nano-emitters
1000 1200 1400 1600
0.0
0.2
0.4
0.6
0.8
1.0
Norm
alize
d PL
inte
nsity
Wavelength (nm)
SlideAdvantage of Near-infrared Region Imaging 2
Nat. Nanotechnol. 2009, 4, 710
- Biomolecules have lower absorption and scattering in the NIR region.
- The NIR optical window can maximize the tissue penetration depth.
<Tissue penetration depth of lights><Effective attenuation coefficientsof biomolecules>
First optical window (FOW; 700 – 900 nm)Second optical window (SOW; 1000 – 1400 nm)
SlideImaging Setup for NIR Fluorescence Multiplexed Imaging 3
InGaAs CCD
ZoomLens
ColorCCD
1000 LPF : 1000 nm long pass filter(open channel)
1050 BPF : 1050 nm band pass filter(short wavelength channel)
1250 LPF : 1250 nm long pass filter(long wavelength channel)
Dichroicmirror
Motorized filter wheel
1000 LPF
1050 BPF1250 LPF
blue arrow: visible light red arrow: infrared light
rotation
Adv. Healthcare Mater. 2018, 7, 1800695.
SlidePbS/CdS QDs for Multiplexed Imaging 4
1000 1200 14000.0
0.2
0.4
0.6
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1.0
Norm
alzie
d FL
inte
nsity
(a.u
.)
Wavelength (nm)
1080-PQD 1280-PQD
20 nm
20 nm
Fabrication of polymer-encapsulated QDs (PQDs)
PMAO-PEG : poly(maleic anhydride-alt-1-octadecene) conjugated with poly(ethylene glycol)
1080-QD 1280-QD
TEM images PbS/CdS QDs for multiplexed imaging Normalized FL spectra of two PQDs
Adv. Healthcare Mater. 2018, 7, 1800695.
SlidePolymer-encapsulated QDs (PQDs) 5
0 2 4 6 8 100
20
40
60
80
100
120
PQD in DMEM w/ 10% FBS at 25 °C PQD in DMEM w/ 10% FBS at 37 °CRe
lative
FL
inten
sity (
%)
Time (day)
10 20 30 400
10
20
30
popu
latio
n (%
)
hydrodynamic size (nm)
1080-PQD 1280-PQD
Relative FL intensity and HD size change over time for PQDs in water
Relative FL intensity change over time for PQDs in cell growth media
Dynamic light scattering histogram of the hydrodynamic (HD) size of polymer-encapsulated QDs (PQDs).
1080-PQD and 1280PQD show the same hydrodynamic sizeand the same Zeta potential.
Adv. Healthcare Mater. 2018, 7, 1800695.
SlideNIR Fluorescence Multiplex Imaging 6
1080-PQD
1080
-PQ
D +
1280
-PQ
D blankagar gel
1280
-PQ
D
S-channel L-channel merged image
O-channelS-channel L-channel1080-PQD 1280-PQD
PQD aqueous solutions
S-channel; 1050 nm band pass filterL-channel; 1250 nm long pass filterO-channel; 1000 nm long pass filter
Nude mouse that was subcutaneously injected agar gel-PQD mixtures
Adv. Healthcare Mater. 2018, 7, 1800695.
SlideBioconjugation of PQDs 7
100 μm
100 μm 100 μm
100 μm 100 μm 100 μm
100 μm100 μm
FA-P
QD
PQD
Human dermal fibroblast cell(folate receptor-negative)
HeLa (human cervical cancer) cell(folate receptor-positive)
PQD : polymer-encapsulated QDFA-PQD : folic acid-conjugated PQD
• 300 nM FA-PQDs or unconjugated PQDs were co-incubated with cells for 8 h. • FA-PQDs can specifically target and label cancer cells that overexpress folate receptors.
Adv. Healthcare Mater. 2018, 7, 1800695.
Slide
The mouse was intravenously
injected with a mixture of two
color NIR-II probes: 1080-PQD
and folic acid-conjugated 1280-
PQD (FA-1280-PQD). NIR-II FL
images under L-channel for FA-
1280-PQD signals. The FL
images were taken 5 min after
the injection.
Whole body in vivo NIR-II image
Adv. Healthcare Mater. 2018, 7, 1800695.
SlideIn vivo Multiplexed NIR-II Imaging 9
tumornormal
L-channelS-channel220
30
125
1080-PQD FA-1280-PQD2.0
2.5
3.0
Tum
or /
Norm
al
The FL signal ratio of tumor region to normal region for 1080-PQD and FA-1280-PQD
(taken 140 min after the injection)
S-channel; 1050 nm band pass filterL-channel; 1250 nm long pass filter
• This NIR-II whole body imaging with the two PQDs provided precise evaluation of active ligand-assisted tumor-targeting of the folic acid conjugated PQDs that was unmixed from permeation and retention effects in tumors that are typically heavily dependent on the hydrodynamic size and surface properties.
Adv. Healthcare Mater. 2018, 7, 1800695.
Unconjugated1080-PQD
FA-Conjugated1280-PQD
Slide10Switching Quantum Dot (QD) Fluorescence
Attaching a switch onto a QD,
thus making the QD-Switch
conjugate can be turned on and off
responding to external stimuli:
light, analyte concentrations,
(pH, ions, etc), enzymatic activities,
and binding events
(small molecule or antigen binding).
Applications for sensors, in vivo
probes, imaging, memory, etc.
SlideActivatable fluorescent probes 11
Bremer, C.; Tung, C. H.; Weissleder, R. Nat. Med. 2001, 7, 743.Lee, S.; Park, K.; Kim, K.; Choi, K.; Kwon, I. C. Chem. Commun. 2008, 4250.
Activatable fluorescent probe : fluorophore whose signal is amplified by the biological event of interests such as enzymatic activity, pH, nucleic acids
event of interest(ex) protease activity,
pH, nucleic acids)
EmitterQuencher
Energy/charge transfer
Simple scheme of activatable fluorescent probeLinker
• Sensitive detection of protein activity, nucleic acid, pH in in vitro and in vivo
with low background signal
• Activatable NIR-II QDs were not reported yet
off-state on-state
SlideDesign of Matrix Metalloproteinase(MMP)-activatable
probe for cancer-microenvironment detection 12
hv
e-
h+
Quenched Photoluminescence Activated Photoluminescence
CB
hv
VB
Peptide cleavage by MMP-2
Quencher
MMP-cleavable peptide sequence
electron transfer
S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
SlideQuenching via photoinduced electron transfer by methylene blue 13
400 600 800 1000 1200 1400 1600
fluorescence intensity (a.u.)
abso
rban
cewavelength (nm)
MB PbS/CdS/ZnS QD
no spectral overlap between QD and MBà no change of FRET based quench
Absorption spectrum of MB andfluorescence spectrum of QD
Methylene blue (MB) :
Energy level diagram of PbS/CdS/ZnS QD and MB
FRET = Foster resonance energy transfer
Fluorescence quench via electron transfer was expected
CB = conduction bandVB = valence band
SlideSynthesis of NIR-II emitting PbS/CdS/ZnS QD 14
thermolysis
zinc oleate, sulfurPbS
CdS
ZnS
PbS/CdS/ZnS
core/shell/shell QD
cation exchangePbS
CdS
PbS/CdS
core/shell QD
cadmium oleate
PbS
PbS QD
lead oleate
sulfur thermolysis
PbS CdS ZnS
conduction band
valence band
Energy
Energy level diagram of QD
Scheme for the fabrication of PbS/CdS/ZnS multishell QD
• Enhanced quantum yield and photostability rather than PbS QDs
SlideHAADF EM Image 15
(a) STEM-HAADF image of PbS/CdS/ZnS QDs. (b) MagnifiedSTEM-HAADF image of single PbS/CdS/ZnS QD.
20 nm
a
PbS core
CdS shell
5 nm
b
STEM : Scanning transmission electron microscopyHAADF : High-angle annular dark-field imaging
S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
Slide16
: oleic acid
ligand exchange
: dihydrolipoic acid,
Ligand exchange from hydrophobic to hydrophilic QD
0 5 10 15 20 25 30 35 400
10
20
30
popu
latio
n (%
)
hydrodynamic size (nm)
average size = 9.7 nm
Hydrodynamic size of PbS/CdS/ZnS QD Color (left) and FL (right) image ofwater-soluble PbS/CdS/ZnS QD
Water-soluble PbS/CdS/ZnS QDs
QD QD
S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
SlideSurface modification for activatable probe 17
Step 1: Maleimide coupling of methylene blue and MMPCP
Step 2: Conjugation of MMPCP-MB with QD
MMP-cleavable peptide sequence (MMCP)
+≡
Cleavage Site
PEG8
D4-
MB+
NH2≡
maleimide-MB
S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
SlideQuenching and activation of QD-MMPCP-MB complex 18
0 10 20 30 40 50 60
1.0
1.5
2.0
2.5
3.0 0 mg/mL 30 mg/mL 10 mg/mL 10 mg/mL+MMP-I 20 mg/mL
rela
tive
FL in
tens
ity
time (min)
Time-dependent FL recovery ofQD-PEG-(-)MMPCP-MB with MMP-2 concentration
• Fluorescence activation was proportional to the concentration of MMP-2• Suppressed activation with MMP inhibitor showed the origin of
FL activation comes from the cleavage activity of MMP-2
100 nM QD-(-)MMPCP-MB solution ([MB]/[QD]=40)
buffer condition : 20 mM Tris, 0.1 mM Ca(NO3)2, 20 μM Zn(NO3)2, 100 mM NaClMMP-I : global MMP inhibitor
S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
SlideHow to design the quencher peptide sequence
1. spacer sequence 19
QD-(-)MMPCP-MBforbidden proteolysis by MMP-2
QD-PEG-(-)MMPCP-MBallowed proteolysis by MMP-2
PEG = polyethylene glycol
0 10 20 30 40 50 601.0
1.5
2.0
2.5
rela
tive
FL in
eten
sity
time (min)
QD-(-)MMPCP-MB QD-PEG-(-)MMPCP-MB
100 nM QD-MMPCP-MB buffered solutionMMP-2 enzyme 20 μg/mL
PbS
CdSZnS
CleavageSite D4-
MB+
PbS
CdSZnS
CleavageSite
PEG8
D4-
MB+
S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
SlideHow to design the quencher peptide sequence
2. charged state of quencher sequence 20
repulsive force
no electrostatic force
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1.0
1.1
1.2
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1.4
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rela
tive
fluoe
rsce
nce
inte
nsity
time (min)
(+) (+/-) (-)
0 5 10 15 200.0
0.2
0.4
0.6
0.8
1.0 (+) (+/-) (-)
rela
tive
PLQ
Y
[MMPCP-MB]/[QD]FL intensity of QD-MMPCP-MB
FL recovery of QD-MMPCP-MB with enzyme
PbS
CdSZnS
CleavageSite
PEG8
Dn-
MB+
After enzymatic cleavageD4-
MB+
D2-
MB+no D
MB+
attractive force
(+) charge
(-) charge
(+/-) charge(net zero charge)
S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
Slideex vivo fluorescence cancer imaging
using NIR-II activatable probe 21
AOM : azoxymethaneDSS : dextran sulfate sodium salt
• colorectal cancer model (AOM/DSS-treated mouse) is known for high upregulation of MMPs in cancer microenvironment
Scheme for ex vivo fluorescence imaging of colon cancer model
SlideColon cancer imaging with activatable probe 22
Time-dependent signal activation
Cancer microenvironment-specific fluorescence activation
Probe : 1 μM QD-PEG-(-)MMPCP-MB in PBS buffer at pH 7.4([MB]/[QD]=40)
excited by 910 nm laser with 200 mW/cm2
exposure time = 90 ms
Time-dependent fluorescence image
S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
SlideNormal colon imaging with activatable probes 23
Time-dependent signal activation
No noticeable fluorescence activation
Time-dependent fluorescence image
Probe : 1 μM QD-PEG-(-)MMPCP-MB in PBS buffer at pH 7.4([MB]/[QD]=40)
excited by 910 nm laser with 200 mW/cm2
exposure time = 90 ms
0 5 10 15 20 25 30 350.5
1.0
1.5
2.0
2.5
3.0
3.5
rela
tive
FL in
tens
ity
time after probe spray (min)
A1 A3 A6 A2 A5
S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
SlideColon cancer imaging with non-activatable probes 24
Time-dependent signal activation
non-activatable probe = QD without MMPCP-MB
Time-dependent fluorescence image
Probe : 1 μM QD in PBS buffer at pH 7.4 excited by 910 nm laser with 200 mW/cm2
exposure time = 90 ms
0 5 10 15 20 25 30 350.5
1.0
1.5
2.0
2.5
3.0
3.5
rela
tive
FL in
tens
ity
time after probe spray (min)
N T3 T1 T4 T2 T5
S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
SlideAcknowledgement 25
Alumni: Junhyuck, Park, Sungwook Jung, Sanghwa Jeong
Group members: Yebin Jung, Youngju Kwon, Junhwa Lee, Wonsuk Lee, Yunmo Sung, Jihye
Lee, Woojin Lee, Eunjae Lee, Seunghwa Hong, Sungbin Yang, Jeongmin Kim, Soomin Lee,
Doowon Choi, Sujin Lee, Anastasia Agnes, Eunjeong Kim
Nanophotonics and Nanomedical Research Group
Collaborator (partial list):
Prof. Nam Ki Lee, SNU
Prof. Jong Bong Lee, POSTECH
Prof. Seung-Jae Myung, AMC
Prof. Chan Ki Pack, AMC
Prof. G-One Ahn, POSTECH
Prof. Junsang Doh, POSTECH
Prof. Jung-Joon Min, Chonnam Univ.
Prof. Chulhong Kim, POSTECH
Prof. Ki Hean Kim, POSTECH
Prof. Euiheon Chung, GIST
Slide
Thank you for listening.
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