概要信息：

REVIEW ARTICLE
nature materials | VOL 4 | JUNE 2005 | www.nature.com/naturematerials 435
Quantum dot bioconjugates for imaging, 
labelling and sensing
One of the fastest moving and most exciting interfaces of nanotechnology is the use of quantum 
dots (QDs) in biology. The unique optical properties of QDs make them appealing as in vivo and 
in vitro fl uorophores in a variety of biological investigations, in which traditional fl uorescent labels 
based on organic molecules fall short of providing long-term stability and simultaneous detection of 
multiple signals. The ability to make QDs water soluble and target them to specifi c biomolecules has 
led to promising applications in cellular labelling, deep-tissue imaging, assay labelling and as effi cient 
fl uorescence resonance energy transfer donors. Despite recent progress, much work still needs to be 
done to achieve reproducible and robust surface functionalization and develop fl exible bioconjugation 
techniques. In this review, we look at current methods for preparing QD bioconjugates as well as 
presenting an overview of applications. The potential of QDs in biology has just begun to be realized 
and new avenues will arise as our ability to manipulate these materials improves.
IGOR L. MEDINTZ1*, H. TETSUO 
UYEDA2, ELLEN R. GOLDMAN1 AND 
HEDI MATTOUSSI2*
1Center for Bio/Molecular Science and Engineering, Code 6900, 
US Naval Research Laboratory, Washington, DC 20375, USA
2Division of Optical Sciences, Code 5611, US Naval Research 
Laboratory, Washington, DC 20375, USA
*e-mail: Imedintz@cbmse.nrl.navy.mil; 
Hedimat@ccs.nrl.navy.mil
One of the fundamental goals in biology is 
to understand the complex spatio–temporal 
interplay of biomolecules from the cellular to 
the integrative level. To study these interactions, 
researchers commonly use fluorescent labelling 
for both in vivo cellular imaging and in vitro 
assay detection1. However, the intrinsic photo-
physical properties of organic and genetically 
encoded fluorophores, which generally have 
broad absorption/emission profiles1 (Fig. 1a) 
and low photobleaching thresholds, have limited 
their effectiveness in long-term imaging and 
‘multiplexing’ (simultaneous detection of multiple 
signals) without complex instrumentation and 
processing2. The seminal publications describing 
the first biological uses of QD-fluorophores 
foresaw that their unique properties could 
overcome these issues3,4. QD properties of interest 
to biologists include high quantum yield, high 
molar extinction coefficients (~10–100× that of 
organic dyes)5,6, broad absorption with narrow, 
symmetric photoluminescence (PL) spectra 
(full-width at half-maximum ~25–40 nm) 
spanning the UV to near-infrared (Fig. 2), large 
effective Stokes shifts (Fig. 1b,c), high resistance 
to photobleaching and exceptional resistance to 
photo- and chemical degradation (Fig. 3a)7–11. 
Compared with molecular dyes, two properties 
in particular stand out: the unparalleled ability 
to size-tune fluorescent emission as a function of 
core size (for binary semiconductor materials), 
and the broad excitation spectra, which allow 
excitation of mixed QD populations at a single 
wavelength far removed (>100 nm) from 
their respective emissions (Fig. 1b,c). QDs 
also display intermittency (blinking) under 
continuous excitation, a property only partially 
understood, and which has been attributed to 
Auger ionization12,13.
SYNTHESIS AND CAPPING STRATEGIES
QDs used in bio-applications are exclusively 
colloidal nanocrystals. The best available QD 
fluorophores for biological applications are made 
of CdSe cores overcoated with a layer of ZnS 
because this chemistry is the most refined. The 
ZnS layer passivates the core surface, protects it 
from oxidation, prevents leeching of the Cd/Se 
into the surrounding solution and also produces a 
substantial improvement in the PL yield5,14. Even 
though thin ZnS (1–2 monolayers) shells often 
nmat1390-print.indd   435 11/5/05   10:18:11 am
Nature Publishing Group© 2005
© 2005 Nature Publishing Group 
 
REVIEW ARTICLE
436 nature materials | VOL 4 | JUNE 2005 | www.nature.com/naturematerials
produce the highest PL yields, thicker ZnS shells 
(4–6 monolayers) provide more core protection 
against oxidation and the harsher conditions 
presented by biological media (for example, acidic 
buffers and cellular organelles). Colloidal QDs 
made of ZnS, CdS, ZnSe, CdTe and PbSe, emitting 
from the UV to the infrared have been prepared14–
29 (Fig. 2); however, these may need refinement 
for bio-applications as issues of reproducible 
synthesis and inorganic passivation remain.
The real breakthrough in synthesizing high-
quality colloidal QDs came when the Bawendi 
group reported24 that use of high-temperature 
growth solvents/ligands (mixture of trioctyl 
phosphine/trioctyl phosphine oxide, TOP/TOPO), 
combined with pyrolysis of organometallic 
precursors, yielded CdSe QDs with highly 
crystalline cores and size distributions of 8–11%. 
The same reaction combined with appropriate 
organometallic precursors was further used to 
1.0
0.5
0.0
1.0
0.5
0.0
1.0
0.5
0.0
1.0
0.5
0.0
Ab
so
rb
an
ce
 (n
or
m
al
iz
ed
)
Ab
so
rb
an
ce
 (n
or
m
al
iz
ed
)
Photolum
inescence (norm
alized)
Photolum
inescence (norm
alized)
Absorption rhodamine red
Absorption DsRed2
Emission rhodamine red
Emission DsRed2
510 nm ABS
510 nm EM
530 nm EM
555 nm EM
570 nm EM
590 nm EM
610 nm EM
400 450 500 550 600 650 700
Wavelength (nm)
rCdSe core (Å)
λmax em. (nm)
13.5
510
14.5
530
17.5
555
19.0
570
21.5
590
24.0
610
b
a
c
d
Figure 1 Comparison of 
rhodamine red/DsRed2 
spectral properties to those 
of QDs highlighting how 
multiple narrow, symmetric QD 
emissions can be used in the 
same spectral window as that 
of an organic or genetically 
encoded dye. a, Absorption 
(Abs) and emission (Em) of 
rhodamine red, a common 
organic dye and genetically 
encoded DsRed2 protein98. 
b, Absorption and emission of 
six different QD dispersions. 
The black line shows the 
absorption of the 510-nm-
emitting QDs. Note that at 
the wavelength of lowest 
absorption for the 510-nm QD, 
~450 nm, the molar extinction 
coeffi cient is greater than 
that of rhodamine red at its 
absorption maxima (~150,000 
versus 129,000 M–1 cm–1). 
c, Photo demonstrating the 
size-tunable fl uorescence 
properties and spectral range 
of the six QD dispersions 
plotted in b versus CdSe core 
size. All samples were excited 
at 365 nm with a UV source. 
For the 610-nm-emitting 
QDs, this translates into a 
Stokes shift of ~250 nm. 
r = radius d, Comparison of 
QD size to a MBP molecule. 
555-nm-emitting CdSe/ZnS 
core/shell QD, diameter ~60 Å, 
surface-functionalized with 
dihydrolipoic acid (red shell 
~9–11 Å) has a diameter 
~78–82 Å. The diagram 
depicts the homogeneous 
orientation MBP assumes 
relative to the QD (Copyright 
National Academy of Sciences 
USA)53. MBP a midsize protein 
(Mr ~ 44 kDa) has dimensions 
of 30 × 40 × 65 Å (ref. 48).
nmat1390-print.indd   436 11/5/05   10:18:14 am
Nature Publishing Group© 2005
© 2005 Nature Publishing Group 
 
REVIEW ARTICLE
nature materials | VOL 4 | JUNE 2005 | www.nature.com/naturematerials 437
overcoat the native CdSe core with a layer of 
wider-bandgap semiconducting material (for 
example, ZnS and CdS)5,14,25. Fine-tuning this 
synthetic scheme highlighted the importance of 
the high-temperature solvent/ligand mixtures, 
along with using less pyrophoric salt precursors 
(CdO and Cd-acetate), for preparing reproducible 
high-quality nanocrystals15,30.
QDs prepared using high-temperature routes 
have no intrinsic aqueous solubility, thus phase-
transfer to aqueous solution requires surface 
functionalization with hydrophilic ligands, either 
through ‘cap exchange’, a process primarily driven 
by mass action, or by encapsulating the original 
nanocrystals in a thick heterofunctional organic 
coating, driven mainly by hydrophobic absorption 
onto the TOP/TOPO-capped QDs. These ligands 
mediate both the colloid’s solubility and serve as 
a point of chemical attachment for biomolecules. 
Capping ligands serve another critical role in 
insulating/passivating/protecting the QD surface 
from deterioration in biological media. Synthesis 
of new caps create an ever-increasing library 
available for obtaining aqueous QD dispersions; 
however, many were created for specialized uses 
and thus have limited general applicability3,14,19,
26,31–38. A representative list of caps and the QD-
dispersal strategies they use is provided in Table 1. 
These strategies can be grouped into three major 
routes. The fi rst uses ‘cap exchange’ and involves 
the substitution of the native TOP/TOPO with 
bifunctional ligands, each presenting a surface-
anchoring moiety to bind to the inorganic QD 
surface (for example, thiol) and an opposing 
hydrophilic end group (for example, hydroxyl, 
carboxyl) to achieve water-compatibility. These 
include an array of thiol and phosphine mono 
and multidentate ligands (Table 1a,b)4,26,41–43. The 
second strategy involves formation of polymerized 
silica shells functionalized with polar groups, 
which insulate the hydrophilic QD (Table 1c)3,40. 
The third method preserves the native TOP/TOPO 
on the QDs and uses variants of amphiphilic 
‘diblock’ and ‘triblock’ copolymers and 
phospholipids to tightly interleave/interdigitate 
the alkylphosphine ligands through hydrophobic 
attraction, whereas the hydrophilic outer 
block permits aqueous dispersion and further 
derivitization (Table 1d,f,g)33–36,44–46.
Is there a place for all these different capping 
strategies? Probably, given that they address the 
requirements of various potential applications. 
But the advantages of each strategy have to be 
carefully weighed against the drawbacks. For 
example, compact mono-mercapto ligands 
(Table 1a), although simple to synthesize, have 
short shelf lives (<1 week) due to dynamic 
thiol–ZnS interactions8. Substitution from mono 
to dithiol dihydrolipoic acid ligands improves 
long-term stability from ~1 week to 1–2 years, 
suggesting that polydentate thiolated ligands 
could be even more effective8,26,43. However, these 
are carboxylic acids and almost all carboxy-
terminated ligands limit dispersion to basic 
pHs8. Silica shells and polymer/phospholipid 
encapsulation provides stability over a broader 
pH range, but result in substantially larger 
hydrophilic QDs. For example, phospholipid 
and block copolymer coatings tend to increase 
the diameter of CdSe–ZnS QDs from ~4–8 nm 
before encapsulation to ~20–30 nm, a size that 
although smaller than most mammalian cells can 
still limit intracellular mobility and may preclude 
Optical coding
Cellular and assay labelling
FRET donor Near-infrared imaging
Photoelectrochemistry
Microbiology-labels
visible400 700
ZnS CdSe
CdTe
PbS
PbSe/TeZnSe
CdS CdSe/Te CdHgTe alloys
Magnetic
FePt
Fe2O3
Co/SmCo5.2
UV < 250 2,500 > Infrared
λ (nm)
Figure 2 Representative QD core materials scaled as a function of their emission wavelength 
superimposed over the spectrum. Representative areas of biological interest are also presented 
corresponding to the pertinent emission highlighting how most biological usage falls in the visible–near 
infrared region. Inset representative materials used for creating magnetic QDs14–29,70.
0 s 20 s 60 s 120 s 180 s
0 s 20 s 60 s 120 s 180 s
a
b
Figure 3 QD resistance to photobleaching and multicolour labelling. a, Top row: Nuclear antigens 
were labelled with QD 630–streptavidin (red), and microtubules were labelled with AlexaFluor 488 
(green) simultaneously in a 3T3 cell. Bottom row: Microtubules were labelled with QD 630–
streptavidin (red), and nuclear antigens were stained green with Alexa 488. Continuous exposure 
times in seconds are indicated (Reprinted by permission of the Nature Publishing Group)34. Note 
the QD resistance to photobleaching under continuous illumination. b, Pseudocoloured image 
depicting fi ve-colour QD staining of fi xed human epithelial cells. Cyan corresponds to 655-nm 
Qdots labelling the nucleus, magenta 605-Qdots labelling Ki-67 protein, orange 525-Qdots 
labelling mitochondria, green 565-Qdots labelling microtubules and red 705-Qdots labelling actin 
fi laments. Courtesy of Quantum Dot Corp.
nmat1390-print.indd   437 11/5/05   10:18:16 am
Nature Publishing Group© 2005
© 2005 Nature Publishing Group 
 
REVIEW ARTICLE
438 nature materials | VOL 4 | JUNE 2005 | www.nature.com/naturematerials
fluorescence resonance energy transfer (FRET)-
based investigations8. Lastly, an issue often 
overlooked in biocompatible QD preparation is 
monitoring of the ligand-exchange efficiency, 
because performing effective cap exchange still 
remains an art form. Clearly needed are systematic 
analytical techniques evaluating the extent of cap 
exchange beyond the final functional test19,46.
INORGANIC–BIOLOGICAL HYBRIDS
Inorganic–biological hybrids are made 
by conjugating inorganic nanostructures 
(nanoparticles, nanorods) with biomolecules 
(proteins, DNAs) and the resulting conjugates 
combine the properties of both materials, 
that is, the spectroscopic characteristics of the 
nanocrystal and the biomolecular function of 
the surface-attached entities. Owing to its finite 
size (comparable to or slightly larger than that 
of many proteins, Fig. 1d) a single QD can be 
conjugated to several proteins simultaneously. 
The QD thus acts as a nanoscaffold for 
attachment of several proteins, or other 
biomolecules, creating a multifunctional 
nanoparticle–biological hybrid. Our work has 
shown that ~15–20 maltose binding proteins 
(MBP, Mr ~ 44 kDa) can be attached 
to each 6-nm-diameter QD (Fig. 1d)48. In 
these assemblies, conjugate dimensions depend 
on key parameters, such as surface cap used and 
number and size of the biomolecules attached to 
the surface. The function is dictated by the 
nature of the biological moieties and their 
conformation; features that have repercussions 
on final performance. Proteins that do not 
have their recognition site exposed away from the 
QD surface may lose their ability to bind a target.
Conjugation schemes for attaching proteins to 
QDs can be divided into three categories, each of 
which has limitations. (i) Use of EDC, 1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide, 
condensation to react carboxy groups on the QD 
surface to amines; (ii) direct binding to the QD 
surface using thiolated peptides or polyhistidine 
(HIS) residues; and (iii) adsorption or non-
covalent self-assembly using engineered proteins. 
Conjugation using EDC condensation applied to 
QDs capped with thiol-alkyl-COOH ligands often 
produce intermediate aggregates due to poor QD 
stability in neutral/acidic buffers. Using this same 
chemistry for QDs encapsulated with polymeric 
shells bearing COOH groups produces large 
conjugates with poor control over the number 
of biomolecules attached to a single nanocrystal. 
Moreover, this chemistry is prone to crosslinking 
and aggregating QDs, because the numerous 
surface functional sites can bind/crosslink the 
numerous protein target sites. Nonetheless, 
this approach was used to prepare commercial 
QD-streptavidin conjugates having ~20 proteins/
conjugate and relatively high quantum yield. 
Streptavidin-coated QDs are used to attach 
additional functionalities to the QDs (mostly 
biotinylated antibodies), however they will bind 
all biotinylated proteins indiscriminately allowing 
only one targeted use.
Direct attachment of proteins/peptides to 
the QD surface is based on two types of QD 
surface–protein interaction; dative thiol-bonding 
between QD surface sulphur atoms and cysteine 
residues19,49 and metal-affi nity coordination 
of HIS residues to the QD surface Zn atoms 
(Table 1h)50,51. The Weiss group demonstrated the 
former by using phytochelatin-related peptides 
to cap CdSe/ZnS core/shell QDs, providing not 
only surface passivation and water solubility, 
but also a point of biochemical modifi cation 
(Table 1h)19. Using peptides for both dispersion 
and biofunctionalization may represent a new class 
of rationally designed multifunctional biological 
cap11,19. By using metal-affi nity coordination, 
HIS-expressing proteins or peptides can be directly 
attached to Zn on the QD-surface. This strong 
interaction (Zn2+-HIS) has a dissociation constant, 
KD, only slightly less than that measured for 6-
HIS to NTA-Ni2+ (10–13) but stronger than most 
antibody bindings (10–6–10–9)51. Functional assays 
with HIS-appended proteins indicate that control 
can be exerted on the fi nal bioconjugate assembly 
through the molar ratios of each participant added 
before self-assembly48,52–54. This strategy allows 
mixed protein surfaces, with the HIS affi nity for 
other metals (Ni, Cu, Co, Fe, Mn) being relevant to 
future materials51.
Engineering proteins to express positively 
charged domains allows them to self-assemble 
onto the surface of negatively charged QDs 
through electrostatic assembly26. This approach 
has proved useful for attaching a variety of 
engineered proteins to QDs including MBP, for 
purification over amylose resin, and Protein G, 
which will bind the IgG portion of an antibody 
thus acting as a linker55. Proteins can also be 
non-specifically adsorbed to QDs56. Regardless 
of the approach used in forming hybrid 
conjugates, three important issues remain: 
(i) the aforementioned lack of conjugation 
strategies; (ii) the lack of homogeneity when 
attaching proteins to QDs; and (iii) the inability 
to finely control ratios of proteins/QD. The 
lack of homogeneous attachment results in 
heterogeneous protein orientation on the QD 
surface, which may produce conjugates with 
surface-attached proteins that are not optimally 
functional. With antibodies, for example, many 
may not be correctly oriented to bind their 
intended target and this will manifest in low 
avidity. It’s worth mentioning that success 
in bioconjugation is intimately tied into 
development of new caps, thus QD success in 
biology will ultimately be driven by cap design.
EXISTING USES OF QUANTUM DOTS IN BIOLOGY
CELLULAR LABELLING
Cellular labelling is where QD use has made the 
most progress and attracted the greatest interest. 
Within the last two years, numerous reports have 
nmat1390-print.indd   438 11/5/05   10:18:17 am
Nature Publishing Group© 2005
© 2005 Nature Publishing Group 
 
REVIEW ARTICLE
nature materials | VOL 4 | JUNE 2005 | www.nature.com/naturematerials 439
S
( (
( (
(
(
(
(
QDs as synthesized5,24,27
ZnS
Core
CdSe
Shell
P O = PS Zn
ZnS
S Zn
TOP TOPO
Soluble in:
toluene
hexanes
chloroform
QD
QD
QD
QD
QD
QD
QD
QD
S P QD S
Hydrophobic Hydrophillic
HS
O
O
O
O H H
OH
Excess ‘cap’
or ligand
–
–
Soluble in:
basic buffer
Biofunctionalization
QD
QD
Biomolecule
(protein/DNA)
Cap–biomolecule linking functionality
Hydrophobic interaction,
disulphide bridge, adsorption,silane
Electrostatic interaction,
disulphide bridge metal affinity, amide bond
QD–Cap linking functionality
Water solubility
-OH
-COOH
-PEG
DNA
O
HO S S
n
HS(CH2)11(OCH2CH2)4OR  R= -H, -CH2COOH
n = 1: mercaptoacetic acid
n = 2,10,15,benzyl
Hydrophillic
S S
S S
S S
S S
 Dative thiol bond
Mercaptocarbonic
acids4,39
Alkylthiol terminated
DNA41
Thioalkylated 
oligo-ethyleneglycols32
Dihydrolipoic acid
derivatives26,43
Mercaptopropyl
silanols3,40
Amine box 
dendrimers31
Bidentate thiols
Silane shell or box dendrimer
Monothiolated caps
SH SH
O
R R = 
-OH
-(OCH2CH2)nOH  n = 3,5,~12
O
R
Two interactions/ligand
HS Si Si RO
O O
R = -SH, -NH2, -PO2CH3
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophillic
Hydrophillic
Hydrophillic
Hydrophillic
H
N
N
H
N
H
H
N
H
N
OH
OH
OH
OH
OH
OH
OH
OH
H
N
N
H
N
H
N
H
N
N
N
HS
OO
O
O
Crosslinked shellSi
Si Si
Si
Si
SiO
O R
O
Si P
S S
Hydrophobic interactions
CH3(CH2)16
CH3(CH2)16
CH3(CH2)14
CH3(CH2)7     NHCO
CH3(CH2)7     NHCO
O
O
O
O
O
O
O
O
O
O
O
O
H P (OCH2CH2)45OCH3
nn
R = Streptavidin
COOH
CONH-R
COOH
n
COOH
CONH-R
COOH
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophobic
Hydrophillic
Hydrophillic
Hydrophillic
Hydrophillic
TOP/TOPO
TOP/TOPO
Phosphatidylethanol
amine
Phosphatidycholine
micelles33
Modified acrylic acid
polymer33,44,45
Poly(maleic anhydride)
alt-1-tetradecene65
Oligomeric phosphines37
S
S
S
P
P
P
R
R
R
RO P P P
RO
RO
O
O O
O
O
O
O OR
O
O
OR
( )n
N
H
R = COX    X = OH: NH-Streptavidin
   
Functionalized oligomeric phosphines
Amphiphilic triblock copolymer *Site for EDC- 
based antibody 
conjugation
COOH
COOH*
CONH-PEG
Amphiphilic triblock 
copolymer46
X
X X
X
XX
X X
R
RR
R
Amphiphilic
saccharides
NH C
O OH
OH
OH
OH
OH
OH
OH
HO
O
OX =     O(CH2)2
Internal alkanes 
interdigitate with TOPO 
Amphiphilic 
saccharides36
O
N
H
H
N
O
O
NH
NH
HNHN
N
N
Zn QD S S
–COOH
–COOH
–NH2–NH2
Direct attachment of protein/peptides to QD surface
(AHHAHHAAD)n
n = 12
H = histidine
Metal-affinity 
coordination
Maltose binding protein-(H)5-COOH
Biotin-G-Cys-E-Cys-G-G-Cys-E-Cys-G-Cha-C-C-Cha-Cmd
H
2 N–protein–COOH
H
2 N–protein–COOH
Dative thiol 
bonding
Cys = cysteine
Cmd = carboxamide
Cha = 3-cyclohexylalanine
Phytochelatin-α-peptides19
Histidine-rich epitopes50
Polyhistidine metal-affinity
coordination51–54
CH2 CCH2CH2 CH2 CH2CH CH C C[ [ [] ] ]
CH 3
CONHC
8 H
17
COOC
4 H
9
COOC
2 H
5
COOH
COOH
R = -(CH2)10CH3
Representative surface-capping strategies Mechanism of interactoin Examples
NHCO
NHCO
zyx
a
b
c
d
e
f
g
h
Table 1 Schematic of generic QD solubilization and biofunctionalization (a–h). Biofunctionalization (second panel from top) uses caps/ligands to provide three functions. 
Linkage to the QD (pink), water solubility (blue) and a biomolecule linking functionality (green). Examples of surface-capping strategies and the mechanism of interaction with 
the QD and the aqueous environment. For the cap exchange (top right) excess thiolated cap displaces the original TOP/TOPO organic coating by binding the ZnS surface with 
the thiol group and imparting hydrophilicity with the charged carboxyl (or other functionalities) yielding water-soluble colloidal QD dispersions.
nmat1390-print.indd   439 11/5/05   10:18:18 am
Nature Publishing Group© 2005
© 2005 Nature Publishing Group 
 
REVIEW ARTICLE
440 nature materials | VOL 4 | JUNE 2005 | www.nature.com/naturematerials
appeared describing the ability of one or more 
‘colour/size’ of biofunctionalized QDs to label 
cells. Many of these reports show that QD labelling 
permits extended visualization of cells under 
continuous illumination as well as multicolour 
imaging, highlighting the advantages offered by 
these fl uorophores (Table 2, Fig. 3)34,56–60. A clear 
differentiation can be made between labelling of live 
or ‘fi xed’ cells (dead, with chemically crosslinked 
components to maintain cellular architecture). 
Fixed cells can be treated ‘harshly’ to facilitate 
entry of the QD reagent by chemically creating 
pores. For labelling live cells, the process must be 
handled judiciously to maintain cellular viability. 
The major hurdle is entry of the relatively large 
QDs into the cell across the cellular membranes’ 
lipid bilayer. Strategies to accomplish this include 
non-specifi c uptake by endocytosis, where QDs 
often end up in endocytic compartments (Table 2); 
direct microinjection of nanolitre volumes, which 
is tedious and limits the number of cells labelled; 
electroporation, which uses charge to physically 
deliver QDs through the membrane and mediated/
targeted uptake61–63. Mediated uptake uses reagents 
such as Lipofectamine 2000, which encapsulates 
QDs within lipid vesicles to facilitate entry into the 
cell63. Targeted uptake exploits the cells’ propensity 
to recognize and internalize QDs labelled with 
specifi c peptides (for example, HIV-derived TAT 
peptide) and even deliver them to specifi c cellular 
compartments such as the nucleus61. Comparison 
of several of these methods showed that the 
Table 2 Representative cellular components and proteins that have been labelled with QDs. *Indicates that labelling was 
performed in live cells.
Cellular component/protein Function
Nucleus/nuclear proteins* Internal organelle containing the genetic information (chromosomal 
DNA), enclosed by a membrane containing pores that mediate 
transport in and out34,61,62. See Fig. 3.
Mitochondria* Organelle providing essential energy-delivery processes, contains its 
own genome62. See Fig. 3.
Microtubules Cytoskeleton protein that maintains cellular structure and reforms 
during movement34. See Fig. 3.
Actin filaments Cytoskeleton protein that maintains cellular structure, helps localize 
other organelles and reforms during movement34. See Fig. 3.
Endocytic compartments* Vesicles that form on cell surfaces and mediate both specific and non-
specific internalization of extracellular molecules56,58.
Mortalin Member of Heat Shock 70 Protein Family. Differential staining pattern 
in normal and precancerous cells59.
Cytokeratin Cytoskeleton protein that is overexpressed and differentially stained in 
many skin cancer cells57.
Cellular membrane proteins and receptors This membrane is a lipid bilayer that encloses the cytoplasm (cellular 
fluid environment) and contains the membrane spanning channels and 
receptors that allow the cell to communicate with its environment.
Serotonin transport proteins* Cell surface transporter of serotonin and related neurotransmitters69.
Prostate-specific membrane antigen* Protein expressed on surface of normal and cancerous prostate cells46.
Her2* Breast cancer marker protein overexpressed on the surface of some 
breast cancer cells34.
Glycine receptors* Main inhibitory neurotransmitter receptor on surface of spinal nerve 
cells66.
erbB/HER* Cellular membrane spanning receptor that mediates cellular response 
to growth factors. Once activated, the receptors undergo endocytosis 
and recycling or degradation. Overexpressed in many cancers67.
p-glycoprotein* Multidrug transporter that spans the membrane and is a mediator of 
multidrug resistance in cancer cells57,58.
nmat1390-print.indd   440 11/5/05   10:18:18 am
Nature Publishing Group© 2005
© 2005 Nature Publishing Group 
 
REVIEW ARTICLE
nature materials | VOL 4 | JUNE 2005 | www.nature.com/naturematerials 441
mediator Lipofectamine 2000 had the highest 
delivery effi ciency, however, the QDs were delivered 
in aggregates62,63. Others combined techniques 
by electroporating QDs covered with a nuclear 
localization sequence into cells and then monitored 
their translocation into the nuclear compartment61. 
Peptide-targeted uptake looks to be the most specifi c 
for delivering dispersed QDs into cells, although 
it may be limited to cells expressing appropriate 
receptors. Once delivered inside the cytoplasm of 
cells, dispersion of the QDs depends strongly on 
their surface coating and pH stability. QDs capped 
with COOH-terminated groups often aggregate 
shortly after introduction, due to their poor stability 
in acidic conditions, whereas protein-coated QDs are 
more dispersed in the cytoplasm.
To demonstrate multicolour labelling of live 
cells, endocytic uptake and selective labelling of 
cell surface proteins were used with antibody-
conjugated QDs, and then QD emission was 
followed for over a week as cellular development 
was monitored, showing that cells tolerate QDs for 
extended periods of time33,58,65. By using antibody-
linked QDs, both the Her2 breast cancer marker 
and specifi c intracellular proteins were labelled in 
both live and fi xed cancer cells, demonstrating that 
QD-probes can cross into cells and specifi cally bind 
their intended intracellular targets34. QDs can also 
be useful markers for tracking cellular movement, 
differentiation and fate33,58,65. Cancer cells seeded 
on top of QDs engulf them and leave behind 
a fl uorescence-free phagokinetic trail65. Using 
several cell lines, the size and shape of trails were 
shown to correlate well with potential invasiveness, 
providing a novel assay for this property65. Micelle-
stabilized QDs injected into Xenopus embryos 
demonstrated cell lineage-tracing during the 
complex developmental process spanning the 
embryo–tadpole stage and equal transfer of QDs 
from mother to daughter cells, again showing long-
term cellular compatibility even with >109 QDs per 
cell33. By exploiting QD photobleaching resistance, 
previously onerous cellular physiology questions 
have been investigated on the single-molecule level. 
These include tracking the diffusion dynamics of 
individual glycine receptors in neuronal cells over 
long periods of time (min), which provided unique 
information on single-protein movement, and 
interestingly, the authors used blinking to identify 
single QDs and differentiate them from aggregates66. 
Additionally, the specifi c erbB/HER cellular 
receptor-mediated fusion and internalization 
process has been observed in single cells67. These 
publications mark the transition from ‘proof-of-
concept’ to useful tool as these complex cellular 
processes could not be tracked continuously with 
photolabile organic fl uorophores.
IN VIVO AND DEEP TISSUE IMAGING
Imaging tissue with far red/near infrared 
excitation overcomes some problems due to 
indigenous tissue autofluorescence, and QD 
spectroscopic properties can be exploited here 
to achieve somewhat deeper penetration than 
the available near-infrared dyes68–70. This was 
demonstrated by synthesizing near-infrared-
emitting QDs (840–860 nm) and applying them to 
sentinel lymph-node mapping in cancer surgery of 
animals70. Using only 5 mW cm–2 excitation, they 
imaged lymph nodes 1 cm deep in tissue, where 
lymphatic vessels were clearly visualized draining 
QD solutions into the sentinel nodes (Fig. 4)70. As 
an added bonus, the location of QD accumulation 
in the excised nodes may be the most likely 
place for the pathologist to find metastatic cells. 
Interestingly, QDs have been demonstrated to 
remain fluorescent in tissues in vivo for four 
months45. In addition to the properties described 
above, QDs have large two-photon cross-sectional 
efficiency with a two-photon fluorescence process 
100–1,000× that of organic dyes. This makes them 
suitable for in vivo deep-tissue imaging using 
two-photon excitation at low intensities63,71. Using 
this technique to excite green emitting QDs in the 
near infrared allowed imaging of mice capillaries 
hundreds of micrometres deep and subcellular 
resolution of mouse brain68,71.
QDs may also be useful for tracking cancer cells 
in vivo during metastasis46,60,63. A multifunctional 
QD probe has been developed46 (Table If) linked 
with tumour-targeting antibodies. In vivo studies in 
mice expressing human cancer showed that these 
QD probes accumulated at the tumour sites. Using a 
slightly different approach, tumour cells were labelled 
with QDs, injected into mice and then tracked with 
multiphoton microscopy as they invaded lung tissue63. 
In both studies, spectral imaging and autofl uorescent 
subtraction allowed multicolour in vivo visualization 
of cells and tissues46,63. The ability to track cells in vivo 
without continuously sacrifi cing animals represents 
a substantial improvement over current techniques. 
1 cm
Colour video Near-infrared fluorescence Colour/near-infrared merge
Au
to
flu
or
es
ce
nc
e
30
 s
ec
 p
os
t-
in
je
ct
io
n
4 
m
in
 
po
st
-in
je
ct
io
n
Im
ag
e-
gu
id
ed
 re
se
ct
io
n
Figure 4 Near-infrared QD 
imaging in vivo. Images of 
the surgical fi eld in a pig 
injected intradermally with 
400 pmol of near-infrared 
QDs in the right groin. Four 
time points are shown from 
top to bottom: before injection 
(autofl uorescence), 30 s after 
injection, 4 min after injection 
and during image-guided 
resection. For each time point, 
colour video (left), near-infrared 
fl uorescence (middle) and 
colour-near infrared merge 
(right) images are shown. Note 
the lymphatic vessel draining 
to the sentinel node from 
the injection site. (Reprinted 
by permission of the Nature 
Publishing Group)70.
nmat1390-print.indd   441 11/5/05   10:18:19 am
Nature Publishing Group© 2005
© 2005 Nature Publishing Group 
 
REVIEW ARTICLE
442 nature materials | VOL 4 | JUNE 2005 | www.nature.com/naturematerials
Although QDs are clearly superior to dyes for these 
purposes, the question of whether the large QD probe 
mirrors true in vivo physiology remains unanswered.
There are many open questions about the 
toxicity of inorganic QDs containing Cd, Se, Zn, 
Te, Hg and Pb72,73. These can be potent toxins, 
neurotoxins and teratogens depending on dosage 
and complexation, accumulating in and damaging 
the liver and nervous system. In small dosages, 
the metals are bound by metallothionein proteins 
and may be excreted slowly or sequestered in 
vivo in adipose and other tissues64,72. Although 
cellular studies have followed QD exposure over 
time33,34,58,60,63, to date there have been no long-
term animal studies assessing QD toxicology. CdSe 
QD toxicity has been examined using a hepatocyte 
culture model and it was found that exposure of 
core CdSe to an oxidative environment caused 
decomposition and desorption of Cd ions, 
whereas adding a shell of 1–2 monolayers of ZnS 
reduced oxidation, but did not fully eliminate 
cytotoxicity induced by 8 h of photooxidation64. 
A rather thick ZnS-overcoating (4–6 monolayers) 
in combination with efficient surface capping/
coating can substantially reduce desorption of 
core ions; nonetheless, extreme radiation can still 
cause desorption of the core ions64. Additionally, it 
has been reported that QDs could damage DNA74 
and that QD surface coatings effect cytotoxicity75. 
Given the broad interest in QDs, these results are 
fuelling a strong debate on this issue64,74,75. The 
fact that some long-term in vivo studies have not 
found evidence of toxicity is promising33,45,58,63, 
but still not a ‘blanket’ endorsement. For in vivo 
imaging, if the metals are safely contained, the 
issue will become one of metabolically clearing 
the nanoparticle, however, hardly anything is 
known about how these particles will be cleared 
by the body72,73. Given the myriad materials, 
preparations and coatings, it is debatable as to 
whether the definitive comprehensive study will 
ever be realized.
QD ASSAY LABELLING
QD assay labelling uses QDs for in vitro 
assay detection of DNA, proteins and other 
biomolecules. DNA-coated QDs have been shown 
as sensitive and specific DNA labels for in situ 
hybridizations47, as probes for human metaphase 
chromosomes76, and in single-nucleotide 
polymorphism and multi-allele DNA detection77. 
Conversely, DNA linked to QD surfaces has been 
used to code and sort the nanocrystals78. These 
results demonstrate that DNA-conjugated QDs 
specifically bind their complements both in 
fixed cells and in vitro. For proteins, our group 
uses a self-assembled electrostatic protein QD-
functionalization strategy to create QD-based 
fluoro-immunoassay reagents. Using antibodies 
conjugated to QDs by an adaptor protein, we have 
carried out multiple demonstrations of analyte 
detection in numerous immunoassays including 
a four-colour multiplex toxin analysis42,79. For 
bioassays, the ability to excite QDs at almost any 
wavelength below the band edge combined with 
high photobleaching resistance and ‘multiplexing’ 
capabilities highlights the unique combination of 
spectral properties that make QD-fluorophores of 
interest to biologist.
QDS AND FRET
As FRET is sensitive to molecular rearrangements 
on the 1–10 m range (a scale correlating to the size 
of biological macromolecules), researchers have 
long used this photophysical process to monitor 
intracellular interactions and binding events1. 
Reports of QDs as FRET donors in a biological 
context appeared quickly39,80,81; however, the full 
potential has only been demonstrated recently. By 
self-assembling acceptor dye-labelled proteins onto 
QD donor surfaces, two unique advantages over 
organic fl uorophores for FRET became apparent: 
QD donor emission could be size-tuned to improve 
spectral overlap with a particular acceptor dye, and 
having several acceptor dyes interact with a single 
QD-donor substantially improved FRET effi ciency 
(Fig. 5a,b)52. The scenario in Fig. 5b demonstrates 
the latter using a 30 Å radius QD with a dye-
labelled protein attached to the QD surface and 
the dye located at 70 Å from the core. Assuming 
a Förster distance (R0) for this QD donor–dye 
acceptor pair of 56 Å, then the FRET effi ciency 
for a single donor–acceptor pair would be 22%. 
By increasing the number of acceptors to fi ve, the 
effi ciency more than doubles to ~58%48,52.
QDs also function as effective protein 
nanoscaffolds and exciton donors for prototype 
self-assembled FRET nanosensors targeting the 
nutrient maltose by using MBP48. QDs could 
even drive biosensors through a two-step FRET 
mechanism overcoming inherent donor–acceptor 
distance limitations, as schematically depicted in 
Fig. 5c, where each 530-nm QD is surrounded by 
~10 MBPs (each mono-labelled with Cy3, one 
shown for clarity)48. β-cyclodextrin-Cy3.5 (β-
CD-Cy3.5) specifi cally bound in the MBP central 
binding pocket completes the QD-10MBP-Cy3-β-
CD-Cy3.5 sensor complex. Excitation of the QD 
results in FRET excitation of the MBP-Cy3, which 
in turn FRET-excites the β-CD-Cy3.5 overcoming 
the low direct QD-Cy3.5 FRET. Added maltose 
displaces β-CD-Cy3.5 leading to increased Cy3 
emission. Using a set of site-specifi cally dye-
labelled MBPs and a FRET strategy analogous to a 
nanoscale global positioning system determination, 
the QD-MBP structure was modelled and the 
results indicate that MBP assumes a homogeneous 
orientation once assembled on the QD surface 
(Fig. 1d)53. This result suggests that it’s possible 
to self-assemble QD–protein conjugates with the 
proteins homogeneously oriented; an important 
fi nding in light of previous concerns about forming 
hybrid structures.
Paradoxically, the excellent QD donor properties 
(long fl uorescent lifetime, broad absorption and high 
extinction coeffi cient) may almost preclude their role 
as FRET acceptors for organic dyes82. The larger size 
of ‘redder’ CdSe and other QDs (emission >600 nm, 
nmat1390-print.indd   442 11/5/05   10:18:20 am
Nature Publishing Group© 2005
© 2005 Nature Publishing Group 
 
REVIEW ARTICLE
nature materials | VOL 4 | JUNE 2005 | www.nature.com/naturematerials 443
Many biological applications of QDs have just begun to be explored. Using combinations of QD emissions 
to create multicolour optical barcodes is promising, as estimates indicate that with just six colours in 10 
different intensities, a library of ~1 million entries could be encoded89. However, this remains ‘proof-
of-concept’ with preliminary reports showing that polystyrene beads optically encoded with QDs and 
targeting DNA can be hybridized and detected at the single bead level89. The Willner group have been 
investigating QD photoelectrochemistry using DNA-driven QD arrays to harness photogenerated 
currents in the pursuit of novel optoelectronic devices90. In an elegant display, CdS QDs were bound in 
the central pocket of a GroEL chaperonin protein complex, which released this cargo on the addition of 
ATP (Fig. B1a,b)91. GroEL is a cylindrical chaperonin protein complex (Mr ~840 kDa, 4.5 nm cavity) that 
functions in refolding denatured proteins in vivo91. The schematic representation shows the formation 
of GroEL–CdS nanoparticle complexes through inclusion of CdS nanoparticles into the cylindrical 
cavity of GroEL, and its ATP-triggered guest release along with transmission electron micrographs of 
these chaperonin–CdS nanoparticle complexes. The models on the sides of the images are schematic 
representations of the end-view of the complexes (Reprinted by permission of the Nature Publishing 
Group)91. This demonstration bodes well for developing a biosensor or intracellular QD sequestering and 
delivery mechanism based on controlled QD release in response to a target analyte. 
The drive to combine the ability to capture, separate and visualize cells (or proteins) within one reagent 
is driving interest in biomagnetic QDs. One approach used antibiotic-coated FePt particles to capture and 
identify bacteria at ultralow concentrations, whereas another used nitriloacetic-acid-coated FePt particles to 
effi ciently capture HIS-appended proteins17,21. In an attempt at hybrid magnetic–luminescent QD complexes, 
polymer-coated Fe2O3 cores overcoated with a CdSe-ZnS QD shell and functionalized with antibodies were 
used to magnetically capture breast cancer cells, which were then viewed fl uorescently92. A unique bis-
functional CdS-FePt luminescent–magnetic dimeric QD particle has been synthesized, however, its biological 
function remains to be explored18. See Fig. B1c for a schematic and high-resolution TEM of the QD-
magnetic CdS-FePt nanoparticles. QDs are also promising tools for visualizing in vitro protein movements, 
such as sliding of actin fi laments93,94, as strain-specifi c microbiological labels95 and for enzymatic monitoring 
of DNA replication96. QDs have even been tested as contrasting agents for fi ngerprint dusting97.
Box 1 Developing technologies
50 nm
5 nm
CdS
nanoparticle
ATP
Mg2+ +K+
Coagulation
Release
GroEL/CdS nanoparticle complex
a
b c CdS-FePt
Figure B1 Novel QD bio-
applications and materials. 
a, GroEL is a cylindrical 
chaperonin protein complex 
(MW ~840 kDa, 4.5 nm 
cavity) that functions 
in refolding denatured 
proteins in vivo91. Schematic 
representation of the 
formation of GroEL–CdS 
nanoparticle complexes by 
inclusion of CdS nanoparticles 
into the cylindrical cavity 
of GroEL, and its ATP-
triggered guest release. 
b, Transmission electron 
micrographs of chaperonin–
CdS nanoparticle complexes. 
The models on the sides of 
the images are schematic 
representations of the 
end-view of the complexes 
(Reprinted by permission 
of the Nature Publishing 
Group)91. c, Schematic 
and high-resolution TEM 
of QD-magnetic CdS–FePt 
nanoparticles (Reprinted with 
permission of the American 
Chemical Society)18.
nmat1390-print.indd   443 11/5/05   10:18:21 am
Nature Publishing Group© 2005
© 2005 Nature Publishing Group 
 
REVIEW ARTICLE
444 nature materials | VOL 4 | JUNE 2005 | www.nature.com/naturematerials
510 510 510 510570 570 570 570
 0.5 ns  2.5 ns  4.5 ns  6.5 ns
nR0
6
nR0
6 + r 6
Eff =
R0 = 56 Å
r (Å) n Eff (%)
70 21.6
58.05
1
70
Protein
Dye
70 Å
30
 Å
QD
R0 = 56 Å
2R0
1.0
0.5
0
Ef
fic
ie
nc
y
56 70
Distance (Å)
530
QD
530
QD
Fret 1 Fret 1
Fret 2Excitation Excitation
Cy3 Cy3
Cy3.5 Cy
3.
5
Emission
Emission
Maltose
MBP
MBP
β-Cyclodextrin-Cy3.5
  Initiation of apoptosis
    DNA degradation
Cancer cell
Antibody
Quantum dot
Quantum dot
Energy transfer
Classical 
photosensitizer
hν
hν
hν
3O2
3O2
3O2
1O2
1O2
Cd2+
Cd2+
ROS
ROS
a
b
c
d
5HIS
5HIS
Figure 5 Properties of QDs as FRET donors. a, Images displaying FRET between a 510-nm-
emitting QD donor and MBP-Cy3 acceptor immediately after a short excitation pulse as recorded 
by the CCD camera at 2-ns intervals. Panel 1: 510-nm QD donor only, Panel 2: MBP-Cy3 
acceptor only, Panel 3: 510-nm QD donor with MBP-Cy3 acceptor self-assembled on the QD 
surface. (Reprinted by permission of the American Chemical Society)52. b, Demonstration of 
improved FRET efficiency (Eff) derived from arraying multiple acceptor dyes around a single 
QD donor acting as a protein scaffold. c, Schematic of a 530QD-MBP-Cy3-β-CD-Cy3.5 maltose 
sensing assembly. (Reprinted by permission of the Nature Publishing Group)48. d, Schematic 
of how QD photosensitizers functionalized with cancer cell-specific antibodies would bind and 
specifically kill a cancer cell in vivo. The antibodies direct the QDs to bind only to cancer cells 
and QDs then harvest either UV or infrared energy (by two-photon excitation) and use this to 
generate reactive oxygen species, ROS, which initiate apoptosis or programmed cell death 
(Figure kindly provided by R. Bakalova)84.
diameter >8 nm) may also preclude FRET as the 
Förster distance (R0, donor/acceptor distance for 50% 
energy transfer) may fall within the core–shell radius, 
suggesting an upper limit on QD size for FRET52,53. 
Beyond sensors, QD-FRET may have potential 
medical usage as photosensitizers in photodynamic 
medical therapy (see Fig. 5d)83,84.
Other potential QD technologies are outlined 
in Box 1.
FUTURE OUTLOOK
Cellular labelling will continue to see substantial 
progress. Studies using ‘extensive’ multiplexing 
(6–10 colours) will focus on elucidating complex 
cellular processes by exploiting QD photobleaching 
resistance and multicolour resolution. Cellular 
events will be studied on the single biomolecule 
level with QDs, although intermittency may become 
an issue for these types of experiments. Work on 
near-infrared dots as tissue probes and contrast 
agents will continue almost exclusively in animal 
models due to unsettled issues of toxicity, which 
will remain a hot issue without a defi nitive near-
term answer. Progress in optical bar-coding will 
encompass some combinatorial chemistry synthesis 
scheme with the QDs functioning as barcodes for the 
synthetic products. Application of QD bar-coding 
to high-throughput or parallel assay formats, such 
as gene-expression monitoring or drug-discovery 
assays, can be anticipated. The newly available 
commercial Qbead and Mosaic Gene Expression 
technology will stimulate use, however, the cost 
of the dedicated support equipment needed for 
decoding may limit broader applications85. An 
interesting derivative of QD-barcodes may include 
magnetic properties for capture. The bifunctional 
magnetic/luminescent QDs are intriguing and 
potentially make an ideal reagent for capturing 
and visualizing low-copy-number biomolecules. 
Biofunctionalized paramagnetic nanocrystals 
targeted to particular in vivo sites, such as tumours, 
may make novel magnetic resonance imaging agents. 
Using bifunctional QDs for concurrent magnetic 
resonance imaging and deep-tissue imaging may 
radically alter in vivo imaging86. FRET-based QD-
biosensors will migrate intracellularly to monitor 
physiological processes in real-time. Interestingly, the 
QD multiplexing potential remains almost untapped 
and so bioassays and studies using more than fi ve 
colours simultaneously can be expected.
Another intriguing area involves QD ‘blinking’ 
or intermittency. Epitaxial QDs do not blink. Recent 
reports suggest that thiol-reducing agents may 
suppress colloidal QD blinking even in biological 
buffers, further suggesting that this process may be 
modulated12,13,87. Understanding of QD blinking 
processes will slowly be elucidated, and harnessing 
this phenomenon may create biosensors that truly 
switch off and on. Use of novel QD rod materials with 
polarized emission may be of benefi t to fl uorescence 
polarization assays88. We can also envisage complex 
QD-bioarrays that incorporate biological functions to 
be fully integrated into nanodevices for light/energy 
harvesting, biosensing or molecular electronics.
nmat1390-print.indd   444 11/5/05   10:18:21 am
Nature Publishing Group© 2005
© 2005 Nature Publishing Group 
 
REVIEW ARTICLE
nature materials | VOL 4 | JUNE 2005 | www.nature.com/naturematerials 445
Will QDs replace fl uorescent dyes? No — rather 
they will complement dye defi ciencies in particular 
applications such as in vivo imaging. Moreover, 
adapting QDs for biological use will teach us critical 
lessons about creating future inorganic–biological 
hybrids with direct relevance to many other materials. 
QDs have far from exhausted their biological 
potential. The shift to enabling everyday research is 
now beginning, mostly driven by cellular labelling. 
Other areas, such as medical imaging, FRET 
biosensing, assay labelling and optical barcoding are 
likely to follow suit.
Note added in proof: While this manuscript was 
in preparation, ref. 99 was published. It provides an 
excellent overview of QD usage for in vivo imaging 
and diagnostics.
doi:10.1038/nmat1390
References
1.  Miyawaki, A. Visualization of the spatial and temporal dynamics of 
intracellular signaling. Dev. Cell 4, 295–305 (2003).
2.  Schrock, E. et al. Multicolor spectral karyotyping of human chromosomes. 
Science 273, 494–497 (1996).
3.  Bruchez, M., Jr, Moronne, M., Gin, P., Weiss, S. & Alivisatos, A. P. 
Semiconductor nanocrystals as fl uorescent biological labels. Science 281, 
2013–2016 (1998).
4.  Chan, W. C. W. & Nie, S. Quantum dot bioconjugates for ultrasensitive 
nonisotopic detection. Science 281, 2016–2018 (1998).
5.  Dabbousi, B. O. et al. (CdSe)ZnS core-shell quantum dots: synthesis and 
optical and structural characterization of a size series of highly luminescent 
materials. J. Phys. Chem. B 101, 9463–9475 (1997).
6.  Leatherdale, C. A., Woo, W. K., Mikulec, F. V. & Bawendi, M. G. On the 
absorption cross section of CdSe nanocrystal quantum dots. J. Phys. Chem. B 
106, 7619–7622 (2002).
7.  Murphy, C. J. Optical sensing with quantum dots. Anal. Chem. 74, 520A–526A 
(2002).
8.  Parak, W. J. et al. Biological applications of colloidal nanocrystals. Nanotech. 
14, R15–R27 (2003).
9.  Niemeyer, C. M. Nanoparticles, proteins, and nucleic acids: biotechnology 
meets materials science. Angew. Chem. Int. Edn Eng. 40, 4128–4158 (2001).
10. Alivisatos, P. The use of nanocrystals in biological detection. Nature Biotechnol. 
22, 47–52 (2004).
11. Mattoussi, H., Kuno, M. K., Goldman, E. R., Anderson, G. P. & Mauro, J. M. in 
Optical Biosensors: Present and Future (eds Ligler, F. S. & Rowe C. A.) 537–569 
(Elsevier, Amsterdam, Netherlands, 2002).
12. Nirmal, M. et al. Fluorescence intermittency in single cadmium selenide 
nanocrystals. Nature 383, 802–806 (1996).
13. Efros, A. L. & Rosen, M. Random telegraph signal in the photoluminescence 
intensity of a single quantum dot. Phys. Rev. Lett. 78, 1110–1113 (1996).
14. Hines, M. A. & Guyot-Sionnest, P. Synthesis and characterization of strongly 
luminescing ZnS-Capped CdSe nanocrystals. J. Phys. Chem. 100, 468–471 (1996).
15. Peng, Z. A. & Peng, X. Formation of high-quality CdTe, CdSe, and CdS 
nanocrystals using CdO as precursor. J. Am. Chem. Soc. 123, 183–184 (2001).
16. Bailey, R. E. & Nie, S. Alloyed semiconductor quantum dots: tuning the 
optical properties without changing the particle size. J. Am. Chem. Soc. 125, 
7100–7106 (2003).
17. Gu, H., Ho, P.-L., Tsang, K. W. T., Wang, L. & Xu, B. Using Biofunctional 
magnetic nanoparticles to capture vancomycin-resistant enterococci and 
other gram-positive bacteria at ultralow concentration. J. Am. Chem. Soc. 125, 
15702–15703 (2003).
18. Gu, H., Zheng, R., Zhang, X. & Xu, B. Facile one-pot synthesis of bifunctional 
heterodimers of nanoparticles: A conjugate of quantum dot and magnetic 
nanoparticles. J. Am. Chem. Soc. 126, 5664–5665 (2004).
19. Pinaud, F., King, D., Moore, H.-P. & Weiss, S. Bioactivation and cell targeting 
of semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides. 
J. Am. Chem. Soc. 126, 6115–6123 (2004).
20. Tsay, J. M., Pfl ughoefft, M., Bentolila, L. A. & Weiss, S. Hybrid approach to the 
synthesis of highly luminescent CdTe/ZnS and CdHgTe/ZnS nanocrystals. J. 
Am. Chem. Soc. 126, 1926–1927 (2004).
21. Xu, C. J. et al. Nitrilotriacetic acid-modifi ed magnetic nanoparticles as a general 
agent to bind histidine-tagged proteins. J. Am. Chem. Soc. 126, 3392–3393 (2004).
22. Murray, C. B., Kagan, C. R. & Bawendi, M. G. Synthesis and characterization 
of monodisperse nanocrystals and close-packed nanocrystal assemblies. Ann. 
Rev. Mater. Sci. 30, 545–610 (2000).
23. Xu, C. J. et al. Dopamine as a robust anchor to immobilize functional 
molecules on the iron oxide shell of magnetic nanoparticles. J. Am. Chem. Soc. 
126, 5664–5665 (2004).
24. Murray, C. B., Norris, D. J. & Bawendi, M. G. Synthesis and characterization 
of nearly monodisperse CdE (E = sulfur, selenium, tellurium) semiconductor 
nanocrystallites. J. Am. Chem. Soc. 115, 8706–8715 (1993).
25. Peng, X., Schlamp, M. C., Kadavanich, A. V. & Alivisatos, A. P. Epitaxial growth 
of highly luminescent CdSe/CdS core/shell nanocrystals with photostability 
and electronic accessibility. J. Am. Chem. Soc. 119, 7019–7029 (1997).
26. Mattoussi, H. et al. Self-assembly of CdSe-ZnS quantum dot bioconjugates using 
an engineered recombinant protein. J. Am. Chem. Soc. 122, 12142–12150 (2000).
27. Hines, M. A. & Guyot-Sionnest, P. Bright UV-blue luminescent colloidal ZnSe 
nanocrystals. J. Phys. Chem. B 102, 3655–3657 (1998).
28. Suyver, J. F., Wuister, S. F., Kelly, J. J. & Meijerink, A. Synthesis and 
photoluminescence nanocrystalline ZnS:Mn2+. Nano Lett. 1, 429–433 (2001).
29. Artmeyev, M. V., Gaponenko, S. V., Germanenko, I. N. & Kapitonov, A. M. 
Irreversible photochemical spectral hole burning in quantum-sized CdS 
nanocrystals embedded in a polymeric fi lm. Chem. Phys. Lett. 243, 450–455 
(1995).
30. Reiss, P., Bleuse, J. & Pron, A. Highly luminescent CdSe/ZnSe core/shell 
nanocrystals of low size dispersion. Nano Lett. 2, 781–784 (2002).
31. Guo, W., Li, J. J., Wang, Y. A. & Peng, X. Conjugation chemistry and 
bioapplications of semiconductor box nanocrystals prepared via dendrimer 
bridging. Chem. Mater. 15, 3125–3133 (2003).
32. Hong, R. et al. Control of protein structure and function through surface 
recognition by tailored nanoparticle scaffolds. J. Am. Chem. Soc. 126, 739–743 
(2004).
33. Dubertret, B. et al. In vivo imaging of quantum dots encapsulated in 
phospholipids micelles. Science 298, 1759–1762 (2002).
34. Wu, X. et al. Immunofl uorescent labeling of cancer marker Her2 and other 
cellular targets with semiconductor quantum dots. Nature Biotechnol. 21, 
41–46 (2003).
35. Pellegrino, T. et al. Hydrophobic nanocrystals coated with an amphiphilic 
polymer shell: A general route to water soluble nanocrystals. Nano Lett. 4, 
703–707 (2004).
36. Osaki, F., Kanamori, T., Sando, S., Sera, T. & Aoyama, Y. A quantum dot 
conjugated sugar ball and its cellular uptake on the size effects of endocytosis 
in the subviral region. J. Am. Chem. Soc. 126, 6520–6521 (2004).
37. Kim, S. & Bawendi, M. G. Oligomeric ligands for luminescent and stable 
nanocrystal quantum dots. J. Am. Chem. Soc. 125, 14652–14653 (2003).
38. Wang, X.-S. et al. Surface passivation of luminescent colloidal quantum dots 
with poly(dimethylaminoethyl methacrylate) through a ligand exchange 
process. J. Am. Chem. Soc. 126, 7784–7785 (2004).
39. Willard, D. M., Carillo, L. L., Jung, J. & Van Orden, A. CdSe-ZnS quantum dots 
as resonance energy transfer donors in a model protein-protein binding assay. 
Nano Lett. 1, 469–474 (2001).
40. Gerion, D. et al. Synthesis and properties of biocompatible water-soluble 
silicacoated CdSe/ZnS semiconductor quantum dots. J. Phys. Chem. B 105, 
8861–8871 (2001).
41. Mitchell, G. P., Mirkin, C. A. & Letsinger, R. L. Programmed assembly of DNA 
functionalized quantum dots. J. Am. Chem. Soc. 121, 8122–8123 (1999).
42. Goldman, E. R. et al. Avidin: a natural bridge for quantum dot-antibody 
conjugates. J. Am. Chem. Soc. 124, 6378–6382 (2002).
43. Uyeda, H. T., Medintz, I, L., Jaiswal, J. K., Simon, S. M. & Mattoussi H. 
Synthesis of compact multidentate ligands to prepare stable hydrophilic 
quantum dot fl uorophores. J. Am. Chem. Soc. 127,3870–3878 (2005).
44. Mattheakis, L. C. et al. Optical coding of mammalian cells using 
semiconductor quantum dots. Anal. Biochem. 327, 200–208 (2004).
45. Ballou, B., Lagerholm, B. C., Ernst, L. A., Bruchez, M. P. & Waggoner, A. S. 
Noninvasive imaging of quantum dots in mice. Bioconj. Chem. 15, 79–86 
(2004).
46. Gao, X., Cui, Y., Levenson, R. M., Chung, L. W. K. & Nie, S. In vivo cancer 
targeting and imaging with semiconductor quantum dots. Nature Biotechnol. 
22, 969–976 (2004).
47. Pathak, S., Choi, S. K., Arnheim, N. & Thompson, M. E. Hydroxylated 
quantum dots as luminescent probes for in situ hybridization. J. Am. Chem. 
Soc. 123, 4103–4104 (2001).
48. Medintz, I. L. et al. Self-assembled nanoscale biosensors based on quantum 
dot FRET donors. Nature Mater. 2, 630–638 (2003).
49. Akerman, M. E., Chan, W. C. W., Laakkonen, P., Bhatia, S. N. & Ruoslahti, E. 
Nanocrystal targeting in vivo. Proc. Natl Acad. Sci. 99, 12617–12621 (2002).
50. Slocik, J. M., Moore, J. T. & Wright, D. W. Monoclonal antibody recognition of 
histidine-rich peptide encapsulated nanoclusters. Nano Lett. 2, 169–173 (2002).
51. Hainfeld, J. F., Liu, W., Halsey, C., M. R., Freimuth, P. & Powell, R., D. Ni-
NTAgold clusters target His-tagged proteins. J. Stuct. Biol. 127, 185–198 
(1999).
52. Clapp, A. R. et al. Fluorescence resonance energy transfer between quantum 
dot donors and dye-labeled protein acceptors. J. Am. Chem. Soc. 126, 301–310 
(2004).
53. Medintz, I., L. et al. A fl uorescence resonance energy transfer derived structure 
of a quantum dot-protein bioconjugate nanoassembly. Proc. Natl Acad. Sci. 
101, 9612–9617 (2004).
54. Medintz, I. L., Trammell, S. A., Mattoussi, H. & Mauro, J. M. Reversible 
modulation of quantum dot photoluminescence using a protein-bound 
photochromic fl uorescence resonance energy transfer acceptor. J. Am. Chem. 
Soc. 126, 30–31 (2004).
nmat1390-print.indd   445 11/5/05   10:18:22 am
Nature Publishing Group© 2005
© 2005 Nature Publishing Group 
 
REVIEW ARTICLE
446 nature materials | VOL 4 | JUNE 2005 | www.nature.com/naturematerials
55. Goldman, E. R. et al. Conjugation of luminescent quantum dots with 
antibodies using an engineered adaptor protein to provide new reagents for 
fl uoroimmunoassays. Anal. Chem. 74, 841–847 (2002).
56. Hanaki, K. et al. Semiconductor quantum dot/albumin complex is a long-life 
and highly photostable endosome marker. Biochem. Biophys. Res. Comm. 302, 
496–501 (2003).
57. Sukhanova, A. et al. Biocompatible fl uorescent nanocrystals for 
immunolabeling of membrane proteins and cells. Anal. Biochem. 324, 60–67 
(2004).
58. Jaiswal, J. K., Mattoussi, H., Mauro, J. M. & Simon, S. M. Long-term multiple 
color imaging of live cells using quantum dot bioconjugates. Nature 
Biotechnol. 21, 47–51 (2003).
59. Kaul, Z. et al. Mortalin imaging in normal and cancer cells with quantum dot 
immuno-conjugates. Cell Res. 13, 503–507 (2003).
60. Hoshino, A., Hanaki, K.-I., Suzuki, K. & Yamamoto, K. Applications of 
Tlymphoma labeled with fl uorescent quantum dots to cell tracing markers in 
mouse body. Biochem. Biophys. Res. Comm. 314, 46–53 (2004).
61. Chen, F. & Gerion, D. Fluorescent CdSe/ZnS nanocrystal-peptide conjugates 
for long-term, nontoxic imaging and nuclear targeting in living cells. Nano 
Lett. 4, 1827–1832 (2004).
62. Derfus, A. M., Chan, W. C. W. & Bhatia, S. N. Intracellular delivery of quantum 
dots for live cell labeling and organelle tracking. Adv. Mater. 16, 961–966 
(2004).
63. Voura, E. B., Jaiswal, J. K., Mattoussi, H. & Simon, S. M. Tracking early 
metastatic progression with quantum dots and emission scanning microscopy. 
Nature Med. 10, 993–998 (2004).
64. Derfus, A. M., Chan, W. C. W. & Bhatia, S. N. Probing the cytotoxicity of 
semiconductor quantum dots. Nano Lett. 4, 11–18 (2004).
65. Pellegrino, T. et al. Quantum dot-based cell motility assay. Differentiation 71, 
542–548 (2003).
66. Dahan, M. et al. Diffusion dynamics of glycine receptors revealed by 
singlequantum dot tracking. Science 302, 442–445 (2003).
67. Lidke, D. S. et al. Quantum dot ligands provide new insights into erbB/HER 
receptor-mediated signal transduction. Nature Biotechnol. 22, 198–203 (2004).
68. Levene, M. J., Dombeck, D. A., Kasischke, K. A., Molloy, R. P. & Webb, W. W. 
In vivo multiphoton microscopy of deep brain tissue. J. Neurophys. 91, 
1908–1912 (2004).
69. Rosenthal, S. J. et al. Targeting cell surface receptors with ligand-conjugated 
nanocrystals. J. Am. Chem. Soc. 124, 4586–4594 (2002).
70. Kim, S. et al. Near-infrared fl uorescent type II quantum dots for sentinel 
lymph node mapping. Nature Biotechnol. 22, 93–97 (2004).
71. Larson, D. R. et al. Water-soluble quantum dots for multiphoton fl uorescence 
imaging in vivo. Science 300, 1434–1437 (2003).
72. Colvin, V. L. The potential environmental impact of engineered nanomaterials. 
Nature Biotechnol. 21, 1166–1170 (2003).
73. Hoet, P. H., Bruske-Hohlfeld, I. & Salata, O. V. Nanoparticles - known and 
unknown health risks. J. Nanobiotechnol. 2, 2–12 (2004).
74. Green, M. & Howman, E. Semiconductor quantum dots and free radical 
induced DNA nicking. Chem. Comm. 121–123 (2005).
75. Kirchner, C. et al. Cytotoxicity of colloidal CdSe and CdSe/ZnS nanoparticles. 
Nano Lett. 5, 331–338 (2005).
76. Xiao, Y. & Barker, P. E. Semiconductor nanocrystal probes for human 
metaphase chromosomes. Nucl. Acids Res. 32, 3 e28 (2004).
77. Gerion, D. et al. Room-temperature single-nucleotide polymorphism and 
multiallele DNA detection using fl uorescent nanocrystals and microarrays. 
Anal. Chem. 75, 4766–4772 (2003).
78. Gerion, D. et al. Sorting fl uorescent nanocrystals with DNA. J. Am. Chem. Soc. 
124, 7070–7074 (2002).
79. Goldman, E. R. et al. Multiplexed toxin analysis using four colors of quantum 
dot fl uororeagents. Anal. Chem. 76, 684–688 (2004).
80. Mamedova, N. N., Kotov, N. A., Rogach, A. L. & Studer, J. Albumin-CdTe 
nanoparticle bioconjugates: preparation, structure, and interunit energy 
transfer with antenna effect. Nano Lett. 1, 281–286 (2001).
81. Tran, P. T., Goldman, E. R., Anderson, G. P., Mauro, J. M., & Mattoussi, H. Use 
of luminescent CdSe-ZnS nanocrystal bioconjugates in quantum dot-based 
nanosensors. Phys. Status Solidi B 229, 427–432 (2002).
82. Clapp, A. R., Medintz, I. L., Fisher, B. R., Anderson, G. P. & Mattoussi, H. Can 
luminescent quantum dots be effi cient energy acceptors with organic dye 
donors. J. Am. Chem. Soc. 127, 1242–1250 (2005).
83. Samia, A. C. S., Chen, X. & Burda, C. Semiconductor quantum dots for 
photodynamic therapy. J. Am. Chem. Soc. 125, 15736–15737 (2003).
84. Bakalova, R., Ohba, H., Zhelev, Z., Ishikawa, M. & Baba, Y. Quantum dots as 
photosensitizers. Nature Biotechnol. 22, 1360–1361 (2004).
85. Xu, H. X. et al. Multiplexed SNP genotyping using the Qbead (TM) system: 
a quantum dot-encoded microsphere-based assay. Nucl. Acids Res. 31, 8 e43 
(2003).
86. Josephson, L., Kircher, M. F., Mahmood, U., Tang, Y. & Weissleder, R. 
Nearinfrared fl uorescent nanoparticles as combined MR/optical imaging 
probes. Bioconj. Chem. 13, 554–560 (2002).
87. Hohng, S. & Ha, T. Near-complete suppression of quantum dot blinking in 
ambient conditions. J. Am. Chem. Soc. 126, 1324–1325 (2004).
88. Hu, J. et al. Linearly polarized emission from colloidal semiconductor 
quantum rods. Science 292, 2060–2063 (2001).
89. Han, M., Gao, X., Su, J. Z. & Nie, S. Quantum-dot-tagged microbeads for 
multiplexed optical coding of biomolecules. Nature Biotechnol. 19, 631–635 
(2001).
90. Katz, E. & Willner, I. Integrated nanoparticle-biomolecule hybrid systems:
synthesis, properties, applications. Angew. Chem. Int. Edn Eng. 43, 6042–6108 
(2004).
91. Ishii, D. et al. Chaperonin-mediated stabilization and ATP-triggered release of 
semiconductor nanoparticles. Nature 423, 628–632 (2003).
92. Wang, D., He, J., Rosenzweig, N. & Rosenzweig, Z. Superparamagnetic Fe2O3 
beads-CdSe/ZnS quantum dots core-shell nanocomposite particles for cell 
separation. Nano Lett. 4, 409–413 (2004).
93. Mansson, A. et al. In vitro sliding of actin fi laments labelled with single 
quantum dots. Biochem. Biophys. Res. Comm. 314, 529–534 (2004).
94. Bachand, G. D. et al. Assembly and transport of nanocrystal CdSe quantum 
dot nanocomposites using microtubules and kinesin motor proteins. Nano 
Lett. 4, 817–821 (2004).
95. Kloepfer, J. A. et al. Quantum dots as strain- and metabolism-specifi c 
microbiological labels. App. Environ. Microbiol. 69, 4205–4213 (2003).
96. Patolsky, F. et al. Lighting-up the dynamics of telomerization and DNA replication 
by CdSe-ZnS quantum dots. J. Am. Chem. Soc. 125, 13918–13919 (2003).
97. Menzel, E. R. et al. Photoluminescent semiconductor nanocrystals for 
fi ngerprint detection. J. Forens. Sci. 45, 545–551 (2000).
98. Baird, G. S., Zacharias, D. A. & Tsien, R. Y. Biochemistry, mutagenesis, and 
oligomerization of DsRed, a red fl uorescent protein from coral. Proc. Natl 
Acad. Sci. 97, 11984–11989 (2000).
99. Michalet, X. et al. Quantum dots for live cells, in vivo imaging, and 
diagnostics. Science 307, 538–544 (2005).
Acknowledgements
The authors acknowledge the Naval Research Laboratory (NRL) and A. Ervin and 
L. Chrisey at the Offi ce of Naval Research (ONR grant N001404WX20270) and A. 
Krishan at DARPA for support. I.L.M. was and H.T.U. is supported by a National 
Research Council Fellowship through NRL.
Correspondence should be addressed to I.M. or H.M.
Competing interests statement
The authors declare that they have no competing fi nancial interests.
nmat1390-print.indd   446 11/5/05   10:18:23 am
Nature Publishing Group© 2005
© 2005 Nature Publishing Group