Quantum Dots – Promise to practice
Bala S. Manian
www.qdots.com
What are Quantum Dots




Nanocrystals with extraordinary Optical
Properties
Like a molecular scale “LED”
Engineer-able colors (infrared to blue)
Compelling advantages over dyes
– One color excitation
– Large stokes shift
– Highly stable –no photobleaching
page 2
Physical Properties of Nanocrystals
Qdot nanocrystals are highly fluorescent,
molecular-sized semiconductor crystals
25nm
CdSe
Size:
Tunable from ~2-10 nm (±3%)
Structure: Highly crystalline
page 3
Shells and Brightness
A shell of a higher band-gap material (ZnS),
produces a more stable and brighter structure
S
Zn
S
Zn
S
S
Zn
Zn
S
Zn
Zn
S
S
Zn
Zn
S
S
Zn
Zn
Zn
S
S
Zn
Zn
S
S
S
Zn
Zn
S
S
Zn
S
Zn
Zn
S
S
Zn
Zn
Zn
S
S
Zn
Zn
S
Zn
S
S
Zn
Zn
S
S
Zn
S
Zn
Zn
S
S
Zn
S
Zn
Zn
S
S
Zn
Cd
S
Zn
Se
S
Zn
Zn
S
Zn
Se
Zn
S
Cd
Zn
S
Se
Zn
S
Cd
Zn
Se
Se
Zn
S
Cd
S
Zn
Se
S
Zn
Cd
S
Zn
Se
Zn
S
Cd
Cd
S
Zn
Se
Zn
S
Cd
Zn
Se
Se
Zn
S
Cd
S
Zn
Se
S
Zn
Cd
S
Zn
Se
Zn
S
Cd
Cd
S
Zn
Se
Zn
S
Cd
Zn
Se
Se
Zn
S
Cd
S
Zn
Se
Se
Zn
Cd
S
Zn
Se
Zn
S
Cd
Cd
S
Zn
Se
Zn
S
Cd
Zn
S
Se
Zn
S
Cd
S
Zn
Se
Se
Zn
Cd
S
Zn
Se
S
Zn
Cd
S
Zn
Se
Zn
S
Zn
Zn
S
Se
Zn
S
Zn
Zn
S
S
Zn
Zn
S
Zn
S
S
Zn
Zn
S
Zn
S
S
Zn
Zn
S
S
Zn
S
Zn
Zn
S
S
Zn
S
Zn
Zn
S
S
Zn
Zn
S
Zn
S
S
Zn
Zn
S
S
S
Zn
Zn
S
S
Zn
Zn
Zn
S
S
Zn
Zn
S
S
Zn
Zn
S
S
Zn
Zn
S
S
Zn
S
S
Zn
S
Zn
S
S
Emission Intensity
(Normalized)
Core and Core/Shell Emission Spectra
Core/Shells
160
120
80
Cores
40
0
450
500
550
600
650
Wavelength (nm)
page 4
700
750
S
Fluorescence
Band gap
Absorption
Valence
Band
Energy levels
Radiationless
decay
Small
Molecules
page 5
Conduction
Band
Qdots® Have a Unique Electronic Structure
Qdots
Semiconductors
Size Dependence Particle in a box
(AU)
Size Dependent Absorbance
and Emission
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
350
5.0 nm
CdSe
2.2 nm
CdSe
450
550
650
Wavelength (nm)
L
Eg ~ 1/L2
page 6
L
Physical Properties of Qdots®
• 100s to 1000s of atoms
• 2-10 nm
page 7
• Highly crystalline
• Properties depend on size
and material
Optical properties of nanocrystals
Ordinary light excites all color quantum dots.
(Any light source “bluer” than the dot of interest works.)
Quantum dots change color with size because additional
energy is required to “confine” the semiconductor
excitation to a smaller volume.
page 8
Time controls size & size distribution
X. G. Peng, J. Wickham, and A.. Alivisatos, J. Am.Chem. Soc. 120, pp. 5343-5344, 1998.
Absorbance
Photoluminescence
Time
(mins)
Std.Dev(%)
240
injection
injection
4
14
210
6
190
55
4
35
12
2
6
0 80 180
Time (minutes)
1
0.2
0
415 500 620
Wavelength (nm)
page 9
Normalized PL Intensity
6
Absorbance (a.u.)
Ave. Size (nm)
8
475
565
690
Wavelength (nm)
Some problems with dye molecules in biology
1
Photochemical
Instability
Intensity
0.8
1
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
0
30
60
90
120
150
180
0
350
Red tail
400
O
O
O
450
500
550
N
O
Unique chemistries
OH
Fluorescein
page 10
600
wavelength (nm)
Time (sec)
HO
“Broad”
Narrow
Texas Red
+
N
650
Dyes Vs Qdots
Fluorescein
Narrow excitation
Red tail
Broad emission
e ~ 80,000 M-1cm-1
Qdots®
Broad excitation
Symmetric emission
Narrow emission
e500 ~ 80,000 M-1cm-1
e350 ~ 400,000 M-1cm-1
page 11
Size- and Material-Dependent
Optical Properties
CdTe
CdSe
Normalized Intensity
ZnSe
350
400
450
500
550
600
650
700
750
Emission Wavelength (nm)
Excitation: ZnSe @ 290 nm, others 365 nm
Material band-gap determines the emission range;
particle size tunes the emission within the range
Nanocrystal quantum yields are as high as 80%
Narrow, symmetric emission spectra minimize
overlap of adjacent colors
page 12
Size Scale
page 13
What is the “Promise”

Many ‘sharp’ colors
– Enables highly multiplexed analysis
– Enables complex color bar-coding

Brightness
– High quantum yield

Photostability
– Integrate for longer to improve sensitivity
– Much easier to use

Size
– Small enough to have minimal interference with biological binding
events

Single wavelength excitation
– Simplified instrumentation
page 14
Background of Quantum Dot Corp.




Founded October, 1998
49 employees, 35 technical
$38.5 million raised to date from VC syndicate
Founding scientists
– Paul Alivisatos, UC Berkeley; Moungi Bawendi, MIT;
Shimon Weiss, LBL (now UCLA)

Research collaborations
– SmithKline Beecham, Genentech, Surromed
– NIH, Carnegie Mellon, Cornell, Vanderbilt, U. Melbourne
– Grants: NIST-ATP, SBIR
page 15
Academic lab to Commercial products
Application
performance
Production
volume
Shelf life/stability
Manufacturing
quality
Cost
page 16
Academic lab
It worked in one test we
published!
It’ll be great in these other 10
things too!
One graduate student can easily
make a batch per week
We use our material pretty much
immediately
We just use what we get.
With a new batch of TOPO, it
takes a while to get things
working again.
Well, the raw materials are very
inexpensive
Commercial product
Works time and again in
customers’ hands.
Demonstrated advantages in
applications.
Sufficient to meet product needs,
including QC.
At least 6 months, and more
depending on application.
Ability to produce material
within application-driven
specifications.
QC – measurement of quality
The labor and capital costs are
within range.
The anatomy of a quantum dot bio-probe
Core Nanocrystal
Inorganic Shell
Capping Group
Organic coating
Functionality
Crosslinker and
Biomolecule
page 17
It is easier said (and published)
than done!
page 18
Areas re-designed and improved
Core synthesis using Cd(II)
v. Cd(0) precursors
New polymer
for water
solubility and
conjugation
Core Nanocrystal
Inorganic Shell
Capping Group
Organic coating
Functionality
Shell chemistry improved photostability
page 19
Crosslinker and
Biomolecule
Different Precursors and Reactions
Organometallic vs. Ionic Cadmium
(CH3)2Cd

Cd0
+ C2H6
Cd2+
+ 2RO-
(evolves gas)

Cd(OR)2

…
+ R3P=Se
Ionic cadmium results in:
Slower nucleation
Faster growth
A scaleable process
A less chaotic process
page 20

CdSe
Cd(0) vs. Cd(II) synthesis
Cd0
CdII
Quantum Yield
35-45%
40-80%
FWHM
~35nm
<25nm
Rxn. Scale (max.)
60ml
at least 1L
Rxn. Time (600nm)
3 hr.
10 min.
Safety
pyrophoric
safe
page 21
Batch reproducibility
Emission Linewidth for Nanocrystals
Synthesized via Two Different Processes
Emission Linewidth
50
45
40
35
30
25
20
Old process
New process
15
10
2000-2001
2001-2002
Batch


page 22
Old process irreproducible (but we didn’t know until we tried)
New process – higher performance and reproducibility
Old v. new nanocrystal morphology
Old Cd(0) core/shells
•6.5 nm diameter
•628 nm, 40 nm FWHM
•35% quantum yield
page 23
New Cd(II) core/shells
5.9 nm/10.9 nm dimensions
611 nm peak emission
22 nm FWHM
72% quantum yield
Inorganic shell chemistry re-developed
Core Nanocrystal
Inorganic Shell
Capping Group
Organic coating
Functionality
Shell chemistry improved photostability
page 24
Crosslinker and
Biomolecule
Dramatic photostability improvements
Normalized Emission
Intensity
Relative Photo-stabilities of Red Core-Shell
Nanocrystals under Deep UV Excitation
1
0.8
0.6
0.4
Standard Shell
Mixed shell
In Water
0.2
0
0
50
100
150
200
Time (s)

page 25
New cores, shells, and water solubilization processes
lead to big improvements in light-initiated decay
Photostability Comparison
Underlying quantum dot stability translates to a large
difference in cell staining stability in a benchmarking test
0 min
1 min
2 min
3 min
2 min
3 min
Texas Red
0 min
1 min
160-84
page 26
Organic coating and functionalization
Core Nanocrystal
New polymer
for water
solubility and
conjugation
Inorganic Shell
Capping Group
Organic coating
Functionality
Crosslinker and
Biomolecule
page 27
New chemistry – AMP strategy
OH
O
octylamine modified
poly-acrylic acid
TOPO coated core-shell
O
OH
O P
NH NH
O
O
O
H
N
O
N
H
H
N
H
N
H
N
OH
O
N
H
N
H
O
O
P
O
OH
O
O P
O
O
O
OH
O
P
P
HN
OH
O
O
HN
O
O
O
O
O
N
H
O
O
NH
O
N
H
H
N
O
O P
HO
NH HN O
O
HO
P
O
OH
NH
P
O
O
O
O
O
H
N
O
P
P
O
O
OH
O
OH
NH
O
HO
O
NH
O
HO
O
P
NH HN O
O
O
HO
P
O
NH NH
O
O
OH
O
P
H
N
O
O
O
OH
H
N
OH
O
O
O NH HN
O
OH
HO
N
H
O
O
HN
HN HN
OH
O
O
O
O
O
HO
O
O P
O
O
P
P
conjugation
adsorption
OH
O
O
O
O
OH
NH HN O
NH NH
O
O
HO
NH
OH
O
H
N
OH
O
OH
O
HO
O
O
N
H
N
H
P
O
N
H
H
N
O
O P
O
O
O
P
NH
NH
O
O
O
O
O
OH
O
cross
link
O
O
O
P
OH
O
O P
O
O
O
O
OH
O
P
P
HN
OH
HO
HO
O
O
O
O
O
HO
O
HO
page 28
O
O
N
H
O
O
O
HO
O
N
H
H
N
O
O P
O
N
H
H
N
H
N
H
N
HO
P
N
H
N
H
O
H
N
HO
O
P
O
OH
O
P
O
O
P
O
O P
P
O
O
O
N
H
O
OH
O
HO
O
P
HO
O
O
O
H
N
O
OH
P
O
OH
O
NH
OH
O
OH
HO
NH
NH HN O
NH
O
O
H
N
O
N
H
H
N
H
N
H
N
O
O
O
O
OH
O
O
OH
O
HN
O
HN HN
O NH HN
O
O
O
HO
O
HN
O
O
H
N
OH
O
OH
O
O
O
OH
HO
HN
N
H
O
O
HN HN
O NH HN
O
O
O
HO
O
OH
Non specific binding improvement
Comparison of materials
AMP-XL-PEG
AMP-XL
AMP
dhla
mua
mpa
maa
0
10
20
30
40
50
60
Counts, thousands
Quantum yield normalized NSB signal
on fixed permeabilized cells
page 29
70
Specific intracellular staining of tubulin
+ SAv-Qdot
nanocrystal
+ Goat anti-rabbit
IgG-biotin
+ SAv-Qdot
nanocrystal
+ Goat anti-rabbit
IgG-biotin
+ Rabbit anti-tubulin
NEGATIVE CONTROL
Control
page 30
SPECIFIC BINDING
Consistent material improvements
Quantum Yield in
Organic/Water (%)
Evolution of Quantum Yield Over Time
80
70
60
50
40
30
20
10
0
1998
1999
2000
2001
2002
2003
year


page 31
Dramatic improvements have been made to all parts
of the quantum dot technology
These have translated into a robust, manufacturable
technology, ready for commercialization
The promise & challenge of nanotechnology

Look at all of these things we can (or could) do.
“Everything is a potential market for
nanotechnology”

BUT…

Can we do any that can not be done any other way?

page 32
Nanotechnology is not a market but it
opens new market opportunities



page 33
Products compete in their specific applications
markets
Nanotechnology can enable new features, improve
efficiency and lower cost.
Can Quantum dots not only compete against existing
application-specific methodologies but give rise to
new ones?
Quantum dot as labels
Incremental advantages

Flow cytometry
– Reduced system complexity
– Multi-parameter labeling
– Antigen density quantitation

Microarray analysis (gene chips)
– Improved sensitivity
– Better reproducibility
– Multiplexing

Fluorescence microscopy market (cell analysis)
– Better labels for confocal microscope
– High sensitivity detection
– Multi-parameter labeling
page 34
Flow cytometry analysis
Saturated intensity of qdots v. Alexa 488 and PerCP
in FACScan cytometer
16000
14000
12000
10000
8000
6000
4000
2000
0
Dye
Qdot now
Qdot anticipated
Green
Red
In collaboration with Bill Hyun, UCSF


page 35
Flow Cytometers are not ideal platform for Qdots
Multi-parameter measurements in simpler single excitation systems is the
attraction.
Static Cytometry
»Laser Scan
Capillary
Sample Application
Capillary with labeled cells
Microarray/genechip analysis
Quantum dot


Cy-3
Improved sensitivity relative to industry-standard Cy3 and Cy5
dyes
Sensitivity gains not realized on most existing scanners
– Opportunity for new instrument platform
page 38
Data courtesy of Mike Bittner, NIH
Fluorescence microscopy analysis
Sav-Alexa488 (100%)
Sav-QdotTM Probe (425%)
Data generated in collaboration with Genentech



page 39
Big improvements in sensitivity, photostability and multianalyte detection ability
Relatively small and fragmented market
But, rapid growth in high information content drug screening
applications
Qdot Spectral Barcodes
Wavelength n( m)
Conventional barcodes line thickness and
spacing give code
Wavelength (
nm )
Qdot spectral codes - emission colors
determine code. Can 'barcode' cells,
beads, …
Code using spectral information, spectral spacing, intensity,etc
page 40
Number of codes increases exponentially
Number of
quantum dot colors
used

page 41
Binary
(absent or
present)
Ternary
(absent, low,
or high)
Quaternary
(absent, low,
medium, high)
1
2
3
4
2
4
9
16
3
8
27
64
4
16
81
256
5
32
243
1024
6
64
729
4096
7
128
2181
16384
Photostability and sensitivity also important attributes
in this application
Quantum dot coded microspheres
10,000 nm = 10 microns
10nm
Different colored Qdot™
nanocrystals attached to
the microsphere.
Red Qdot™ nanocrystal
labeled analyte binds to
its partner on the
microsphere.
Ligand attached to the
microsphere capable of
binding its partner.


page 42
Quantum dot ‘barcodes’ incorporated into beads
Beads used in highly parallel biological assays
The Qbead  system
HTS
Beads
Proteomics
Genotyping
Reader
Platform
The alternative to gene chip technology…
page 43
Gene
expression
Applications
Multiplexed SNP assays (with Glaxo SmithKline)
Frequency of Blind Samples Q2
CYP1A1-T6235C
Frequency of Blind Samples Q1
CYP1A1-A4889G
Frequency of Blind Samples Q4
CYP2C18-T479C
60
35
30
25
20
15
10
5
0
50
40
30
Frequency
20
10
0.
02
5
0.
12
5
0.
22
5
0.
32
5
0.
42
5
0.
52
5
0.
62
5
0.
72
5
0.
82
5
0.
92
5
M
or
e
Frequency of Blind Samples Q7
CYP2C8-C1479T
Frequency of Blind Samples Q8
CYP2C8-G416A
Frequency of Blind Samples Q6
CYP2C8-C1196T
20
15
Frequency
10
5
Frequency
0.
02
5
0.
12
5
0.
22
5
0.
32
5
0.
42
5
0.
52
5
0.
62
5
0.
72
5
0.
82
5
0.
92
5
M
or
e
0.
02
5
0.
12
5
0.
22
5
0.
32
5
0.
42
5
0.
52
5
0.
62
5
0.
72
5
0.
82
5
0.
92
5
M
or
e
0
35
30
25
20
15
10
5
0
Frequency of Blind Samples Q10
CYP2D6-C2938T
45
40
35
30
25
20
15
10
5
0
Frequency Blind Samples Q15
CYP3A5-T(-369)G
Frequency Blind Samples Q13
CYP2D6-G4268C
30
25
60
25
20
50
20
15
40
15
Frequency
Frequency
30
0.
02
5
0.
12
5
0.
22
5
0.
32
5
0.
42
5
0.
52
5
0.
62
5
0.
72
5
0.
82
5
0.
92
5
M
or
e
1
0.85
0.925
0.7
0.775
0.55
0.625
0.4
0
0.475
0
0.25
10
0.325
5
0
0.1
5
0.175
20
0.025
10
0.
02
5
0.
12
5
0.
22
5
0.
32
5
0.
42
5
0.
52
5
0.
62
5
0.
72
5
0.
82
5
0.
92
5
M
or
e
10
page 44
Frequency
0.
02
5
0.
12
5
0.
22
5
0.
32
5
0.
42
5
0.
52
5
0.
62
5
0.
72
5
0.
82
5
0.
92
5
M
or
e
25
Frequency
0.
02
5
0.
12
5
0.
22
5
0.
32
5
0.
42
5
0.
52
5
0.
62
5
0.
72
5
0.
82
5
0.
92
5
M
or
e
1
0.
02
5
0.
1
0.
17
5
0.
25
0.
32
5
0.
4
0.
47
5
0.
55
0.
62
5
0.
7
0.
77
5
0.
85
0.
92
5
0
70
60
50
40
30
20
10
0
Frequency
Multiplexed Assays
Qbead liquid arrays allow for
highly customizable genomic and
proteomic assays
1
2
3
4
Lab in a test tube is the successor to
gene/proteomic chips
page 45
Qcell System Encoding/Detection
Cells can be encoded in the same manner as beads
and then sensitively detected and decoded by
imaging
Encode, Mix, Assay, Decode.
Assays probe cellular functions BEYOND binding
page 46
Qcell Codes
7 Normalized Codes
(4 colors)
Green
Green
529nm
Red & Orange
30
30
25
25
20
20
15
15
10
10
5
Red
Red++
Orange
Orange
5
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
1
18
-5
2
3
4
5
6
Chartreuse
8
9
10
11
12
13
14
15
16
17
18
Red & Chartruese
35
Yellow-Green
569nm
7
-5
20
30
Red
Red++
Chartreuse
Yellow-Green
15
25
20
10
15
10
5
5
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
1
18
-5
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
-5
Orange
Orange & Green
40
25
35
20
Orange
612nm
Orange
Orange++
Green
Green
30
25
15
20
10
15
10
5
5
0
0
-5
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
-5
Red
30
25
Red
630nm
20
15
10
5
0
1
-5
page 47
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
3-plex Ca2+ Assay Using Encoded Cells
CHO cells
(unencoded)
Uninduced
Orange 610 nm M1 Muscarinic
Red 620 nm β2-Adrenergic
Induced
( +Carbachol)
Intensity (Fluo-3)
Calcium Response of Qcell Assay
160
140
120
100
80
60
40
20
0
M1-Muscarinic
B2-Adrenergic
CHO
Cell-type (based on code)
page 48
Many different product opportunities in many different
markets

Quantum dot labels
– Microarray labeling, microscopy, flow cytometry, and others
– In vitro diagnostic tests
– Others…

Qbead system
– Gene expression, protein profiling, SNP analysis, diagnostics.
– Others…

Qcell system
– Drug specificity screening
– Toxicology, others

Non-biological applications
– Security inks and color coding
– Opto-electronics
– Others…
page 49
Promise reduced to Practice


Innovations in the areas of inorganic and organic
chemistry as well as in material science have made
quantum dot application in life science a reality.
Quantum dots use have been demonstrated as:
– Fluorescent labels
– Encoding beads for genomic and proteomic applications
– Encoding cells to permit multiplexed cell function assays
page 50
Where is the Killer apps?
How is this innovation process unfolds?
page 51
Conclusions

Dramatic performance improvements seen over the
past 2 years
– Manufacturing of many quantum dot colors is at required
scale and high quality


page 52
Highly competitive in rapid-growth biological assays
marketplace
Many different business opportunities will allow
significant QDC products, and partnerships leading
to the new and exciting applications
Acknowledgements
Chemistry:
Surface Chemistry Group
Marcel Bruchez, Ed Adams, Bei Li, Jianquan Liu, Thearith
Ung, Yanzheng Xu, Linh Nguyen, Kari Haley, Christopher Ng
Nanocrystal Group
Joe Treadway, Don Zehnder, Jeff Larson, Anh Truong,
Mihai Buretea, Marc Schrier
Biology:
Cellular Imaging Group/Qbead™ Assays
Xingyong Wu, Hongjian Liu, Hugh Daniels / Hongxia Xu,
Mike Sha, Janet Uphoff, Edith Wong
Cell Encoding Group
Larry Mattheakis, Yun-Jung Choi, Jing Gong, Jennifer Diaz
Engineering:
Qbead™ Detection
page 53
Jian Jin, Huayong Yong, Will Molenkamp
Research Collaborations
Pharma:
GlaxoSmithKline, Genentech
Academic:
NIH, Novartis Institute, Carnegie Mellon,
Cornell University, Vanderbilt University
Federal:
NIST-ATP, SBIR
page 54