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2014

Ultra-small fluorescent inorganic nanoparticles for bioimaging

Zhen Li
University of Wollongong, [email protected]

Qiao Sun
University of Queensland

Yian Zhu
University of Queensland

Bien Tan
Huazhong University of Science and Technology

Zhi Ping Xu
University of Queensland

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Recommended Citation
Li, Zhen; Sun, Qiao; Zhu, Yian; Tan, Bien; Xu, Zhi Ping; and Dou, S X., "Ultra-small fluorescent inorganic
nanoparticles for bioimaging" (2014). Australian Institute for Innovative Materials - Papers. 1038.
https://ro.uow.edu.au/aiimpapers/1038

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Ultra-small fluorescent inorganic nanoparticles for bioimaging

Abstract
The novel optical, electrical, and magnetic properties of ultra-small inorganic nanoparticles make them
very attractive in diverse applications in the fields of health, clean and renewable energy, and
environmental sustainability. This article comprehensively summarizes state-of-the-art fluorescence
imaging using ultra-small nanoparticles as probes, including quantum dots, metal nanoclusters, carbon
nanomaterials, up-conversion, and silicon nanomaterials.

Keywords
inorganic, nanoparticles, bioimaging, fluorescent, small, ultra

Disciplines
Engineering | Physical Sciences and Mathematics

Publication Details
Li, Z., Sun, Q., Zhu, Y., Tan, B., Xu, Z. & Dou, S. Xue. (2014). Ultra-small fluorescent inorganic nanoparticles
for bioimaging. Journal of Materials Chemistry B, 2 (19), 2793-2818.

Authors
Zhen Li, Qiao Sun, Yian Zhu, Bien Tan, Zhi Ping Xu, and S X. Dou

This journal article is available at Research Online: https://ro.uow.edu.au/aiimpapers/1038

 Ultra-small fluorescent inorganic nanoparticles for bioimaging
Zhen Li,*a Qiao Sun,b Yian Zhu,b Bien Tan,c Zhi Ping Xu,*b and Shi Xue Doua
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x

5 The novel optical, electrical, and magnetic properties of ultra-small inorganic nanoparticles make them
very attractive in diverse applications in the fields of health, clean and renewable energy, and
environmental sustainability. This article comprehensively summarizes state-of-the-art fluorescence
imaging using ultra-small nanoparticles as probes, including quantum dots, metal nanoclusters, carbon
nanomaterials, up-conversion, and silicon nanomaterials.

10 1 Introduction
When the size of inorganic materials is reduced to the nanoscale photobleaching.9 These properties render them robust for
range, they exhibit unusual optical, electrical, magnetic, 50 fluorescent probes for biolabelling and bioimaging.9-12 In recent
mechanical, and chemical properties, distinctly different from years, other fluorescent nanomaterials, such as ultra-small metal
those in their bulk analogues. For example, semiconductor nanoclusters, fluorescent carbon and graphene dots, up-
15 nanocrystals (usually referred to as quantum dots (QDs)) exhibit conversion nanocrystals, and silicon nanoparticles have been
strong size-dependence of their optical properties when their size exploited as alternatives to conventional QDs. In the following
is smaller than the Bohr exciton radius.1 Magnetic iron oxide 55 sections, we introduce these fluorescent nanomaterials from
nanoparticles become superparamagnetic when their size is viewpoints of preparation and functionalization to satisfy the
reduced below the critical size where they behave as individual requirements for routine labelling and imaging of cells and
20 magnetic domains.2 Carbon nanotubes show remarkable tensile tissues. Advanced applications of fluorescent nanomaterials in
strength,3 and graphene exhibits remarkably high electron living systems as sensors for enzyme, oxygen, metal ions, and
mobility.4 Their novel properties make these nanomaterials very 60 pH, have readily been described elsewhere.13-16
attractive in diverse applications, ranging from energy conversion For bioimaging, fluorescent nanoparticles should have water-
and storage to biomedical imaging. In this article, we summarize solubility, biocompatibility, chemical- and photo-stability. They
25 the recent advances in ultra-small inorganic nanoparticles for should also have uniform size and high quantum yield (QY) for
fluorescence imaging (Table 1), especially those smaller than 10 optimized brightness, narrow and symmetric emission for
nm as they are easily taken up and excreted, and show longer 65 multiplexing and colour saturation, and minimized blinking for
blood circulation time in comparison with larger ones. light output stability. In the second part, we introduce the
For fluorescent materials, there are two kinds of development in synthesis and surface modification of fluorescent
30 photoluminescence mechanisms, i.e. down conversion and up QDs (especially CdSe- and CdTe based II-VI QDs) to result in
conversion.5 The down-conversion process normally absorbs one water-soluble, biocompatible and highly stable QDs with high
high energy photon and emits a low energy photon, e.g. a Stokes- 70 QY, together with their routine bioimaging applications and their
shift emission. In contrast, up-conversion is an anti-Stokes toxicity. In the third section, we describe extremely small metal
process that converts the absorbed low energy light into higher nanoclusters (usually smaller than 2 nm) as an emerging
35 energy emission via multiple absorptions or energy transfer fluorescent probes, and address the difficulties in their synthesis,
processes. The fluorescence generated by both processes has long characterization, modification, and imaging application. In the
been used in molecular imaging to visualize cell biology at many 75 fourth part, we bring in carbon-based fluorescent nanoprobes
levels.6, 7 The first fluorescence imaging could be dated back to including carbon dots and graphene quantum dots (GQDs), which
1924 when Policard observed red fluorescence from endogenous are usually smaller than 10 nm. These carbon-based nanoprobes
40 porphyrins in tumours illuminated with an ultraviolet light.8 Since have excellent biocompatibility and unique properties (e.g. both
then, advances in molecular biology, organic chemistry and up-conversion and down-conversion emissions). In the fifth
material science have revealed several classes of promising 80 section, we briefly introduce lanthanide-based up-conversion
probes for fluorescence imaging, which include small organic nanocrystals, which have attracted considerable attention in
dyes, fluorescent proteins, and fluorescent inorganic recent years. Most of up-conversion nanoprobes have a large size
45 nanoparticles.6 Compared with organic dyes and fluorescent (>10 nm) and are out of the scope of this article. In the sixth part,
proteins, fluorescent inorganic nanoparticles have several distinct we discuss fluorescent silicon nanoparticles, which have excellent
advantages. For example, QDs have high absorbance, high QY, 85 biocompatibility and stability. In the last part, we highlight the
narrow emission, large Stokes shifts, and high resistance to major challenges and perspectives of ultra-small fluorescent
 nanoparticles and fluorescence bioimaging.
Table 1. Comparison of different types of fluorescent inorganic nanoparticles.

Types Representatives Preparation Size (nm) Advantages Disadvantages Applications

QDs CdSe, CdTe, InP, Solvothermal and < 10 Tuneable size and toxicity Fluorescent labels
CuInS2, CuInSe2, hydrothermal fluorescence, and sensors
and their core- methods high QY and
shell relatively stable
nanostructures
Metal NCs Au, Ag, Cu, Pt, Pd Reduction of metal <2 Ultra-small size, sensitive fluorescence, Imaging of cells
salts or etching of easily taken up and difficult for modification and tissues.
large metal excreted. and functionalization Sensors
nanoparticles
Carbon-based C-dots, GQDs Solvothermal and <10 Tunable Instability and unclear of Fluorescent
materials hydrothermal fluorescence, fluorescence mechanism biomarkers and
methods excellent sensors
biocompatibility
UCNs Yb3+/Ln3+-doped Solvothermal and most >10, low background, Low QY, potential toxicity Multimodal
NaYF4, GdYF4, hydrothermal few <10 large anti-Stokes imaging agents and
and their core- methods shifts, sharp drug carriers
shell emission, high
nanostructures stability, deep
penetration
Si NPs Si Etching annealed <5 Small size, Ultrahigh Laborious synthesis, Long-term imaging
SiOx, reduction of stability Low QY, Difficult to tune and labeling
SiCl4, and reaction fluorescence
of Na4Si4 with
NH4Br
QDs: quantum dots; UCNs: up-conversion nanocrystals; NPs: nanoparticles; GQDs: graphene quantum dots; QY: quantum yield

cadmium carboxylate, Cd(OOCR) 2; selenium oxide, SeO2,

2 Semiconducting Fluorescent QDs etc.), non-coordinating solvents (e.g. 1-octadecene, ODE)
5 2.1 Synthesis of monodisperse QDs with high QYs 35 and stabilizers 27 (e.g. octyldiphenylamine (ODPA),
diethylenetriaminepentaacetic acid (DTPA), hexadecyl amine
Fluorescent QDs include semiconducting nanoparticles from
(HDA), etc.) to generate monodisperse QDs with high QY.
Groups IV (Si and Ge dots),17-20 II-VI (CdE and ZnE, E = S, Se,
Monodisperse II-VI QDs with different sizes and shapes can
and Te), III-V (InP), and I-III-VI (CuInS2, CuInSe2),21-23 in which
be obtained by controlling their nucleation and growth processes
the II-VI QDs (especially CdSe and CdTe based QDs) have been
40 through optimization of monomer concentration and reactivity,
10 extensively investigated as prototypes of semiconductor QDs due
molar ratio, reaction temperature, ligands, etc. (Figure 1).28-31 It
to their strong quantum confinement effects and high
has been found that slight modification of reaction parameters
fluorescence QYs. II-VI colloidal fluorescent QDs can be
can lead to a broad variety of particle sizes and shapes. For
prepared in organic solvents or aqueous solutions. Organic routes
example, Peng et al. demonstrated that the size and size
are usually selected to prepare monodisperse and highly
45 distribution of CdSe dots can be manipulated by the monomer
15 fluorescent QDs. Discovered in 1981, QDs did not receive
concentration.32 At high monomer concentrations, the smaller
intensive attention until 1993, when a breakthrough in
nanoparticles grow faster than larger ones, which results in the
preparation of colloidal QDs in solution was achieved.24
size distribution being “focused”. If the monomer concentration
Monodisperse cadmium chalcogenide (CdE, E = S, Se, and/or Te)
drops below a critical threshold, the smaller particles are depleted
QDs were prepared by fast injection of a solution of
50 as larger ones grow (i.e. Ostwald ripening), and the size
20 precursors (organometallic Cd and Se/S/Te dissolved in
distribution gets broader or is “defocused”. Controlling the
trioctylphosphine (TOP)) into a high-boiling-point (~ 300 °C)
nanoparticle growth kinetics can result in a narrow particle size
coordinating solvent trioctylphosphine oxide (TOPO). 24 distribution (5% standard deviation) without the size-
These QDs had a narrow particle size distribution with 10%
selective precipitation. 32
standard deviation, which was reduced to 5% after size-
55 It was observed that the QY increased monotonically to
25 selective precipitation. Their fluorescent QY was about 10%.
the maximum value and then decreased with the growth
The key in this “TOPO-TOP” approach is a burst of
time. 33 Such a photoluminescence bright point indicates an
nucleation which can be effectively separated from the
optimal surface structure/reconstruction. Use of a large Se/Cd
growth process.25 The use of highly flammable and toxic ratio (10/1) can result in very bright CdSe QDs with QY of
dimethylcadmium, however, limited the applicability of this 60 85% at room temperature. The high QY of these QDs is
30 approach at that time. Extensive efforts have been made to
attributed to stabilization of organic ligands on the surface. Since
develop and optimize this approach by using various stable
these ligands can be chemically degraded and detached from the
and low toxicity precursors 26 (e.g. cadmium oxide, CdO;
surface, the photo- and chemical stability of the core is
 by Wang et al. 38 The high fluorescence QY and non-blinking
30 QDs make them very useful in applications requiring a

continuous output of single photons.

During the preparation of QDs, attempts to adjust the growth
kinetics of the QDs incidentally led to the development of one-
dimensional (1D) nanorods.29 By using very high precursor
35 concentrations and a defined admixture of alkylphosphonic acids

and trioctylphosphine oxide (TOPO), 1D and even more complex

structures such as arrows, teardrops, or tetrapods were
Figure 1. Transmission electron microscope (TEM) images synthesized (Figure 1).30 Recently, we demonstrated that doped
and growth paths of CdSe nanocrystals with different and undoped 1D semiconductor nanostructures can be produced
morphology. Reproduced from Ref. 30. 40 by using a lower precursor concentration in the presence of

bismuth nanoparticles.39-44 This is in contrast to the synthesis of

1D nanostructures without nanocatalysts. These nanowires
exhibited unusual optical,45, 46 electronic,47, 48 and magnetic40
properties with potential diverse applications.48, 49 The fast
45 growth process resulted in crystal twinning and defects in the

nanowires,50, 51 leading to a low fluorescence QY (< 1%) which

could be improved by more than three times through coating with
a wide-band-gap shell.52
Despite monodispersity, high QY, and stability, these QDs
50 generated in organic solvents are normally hydrophobic and

have to be modified in order to be water-soluble and

biocompatible for bioapplications. The modification leads to
the decrease in fluorescence QY, e.g. the QY of above
perfect core-shell CdSe@CdS QDs decreased from 94% to
36
55 77% after transferred into PBS solution with PEG-SH.
Figure 2. TEM images and blinking behaviour of core-shell Therefore, direct preparation of QDs in aqueous solution has
CdSe@CdS nanoparticles: (a) 2.2 nm CdSe core with 2.4 nm been developed almost simultaneously.
CdS shell; (b) 2.2 nm CdSe core with 0.7 nm CdS shell.
The aqueous approach was firstly adopted by Henglein et
Reproduced from Ref. 36.
al. to prepare CdS nanoclusters in 1982. 53 They also reported
sometimes severely affected. In order to improve their 60 the first example of the preparation of CdTe QDs in aqueous
luminescence and photostability, wide-band-gap shells (e.g. solution. 54 The resultant CdTe QDs did not show
cadmium sulphide (CdS) and zinc sulphide (ZnS)) have been fluorescence, however. Rogach et al. synthesized stable
coated onto their surface to form core-shell QDs.34, 35 Li et al. fluorescent CdTe QDs with a QY of 3% by using thioglycerol
5 developed a successive ion layer adsorption and reaction and mercaptoethanol as stabilizers. 55 Later on, many efforts
(SILAR) technique to epitaxially grow shells in a non- 65 were made to improve QD fluorescence QY by using
coordinating solvent.34 The resultant core-shell CdSe@CdS QDs different stabilizers (thioglycolic acid (TGA);
had a QY of 40%. Xie and his co-workers further developed mercaptopropionic acid (MPA)), precursor ratios, and
this approach to prepare CdSe-core-multishell QDs with QY manners of heating (hydrothermal and microwave
10 up to 85%. 35 Recently, Chen et al. used cadmium oleate and methods). 56 Under the optimal conditions, the QY of water -
octanethiol as Cd- and S-precursors, and prepared nearly 70 soluble CdTe-based QDs can reach as high as 84%, which is
perfect core-shell CdSe@CdS QDs with the highest QY comparable to that of the above-mentioned hydrophobic QDs.
(97%) ever reported (Figure 2). 36 The slow continuous High fluorescence QY also can be obtained by surface
precursor infusion and the relatively low reactivity of modification of as-synthesized QDs with illumination. For
15 octanethiol provide optimal condition s for passivation of the example, the fluorescence QY of CdTe QDs was drastically
CdSe surface and growth of the CdS shell. Compared with 75 improved from 8% to 85% after 28 -day illumination, due to
conventional core-shell CdSe@CdS QDs, these new QDs the formation of the core-shell structure (i.e. CdTe@CdS)
featured significantly suppressed blinking, with an average with the assistance of illumination. 57
fluorescence on/off time ratio of 94:6 for single large core - The above water-soluble QDs were normally prepared in
20 shell nanocrystals (Figure 2). The blinking was gradually strong basic solution (pH > 8), which limits their bio-
suppressed with increasing shell thickness. 36 In addition to 80 applications, as most biological activities take place under
Wurtzite core-shell CdSe@CdS QDs, zinc-blende core-shell neutral-pH conditions. Adjusting the solution pH to neutral could
analogues with suppressed blinking (>95% on time) were quench the fluorescence of the QDs. Therefore, it is of great
also prepared by Qin and co-workers.37 These zinc blende interest to develop a novel approach for preparing highly
25 core-shell CdSe@CdS QDs exhibited a QY of 90%. It should fluorescent water-soluble QDs from stable precursors under
be noted that non-blinking core-shell CdZnSe@ZnSe QDs, 85 neutral pH conditions. Recently, we have successfully
which exhibited complete suppression of blinking on the time synthesized highly fluorescent (84% QY) mercaptosuccinic acid
scale from milliseconds to hours, were successfully prepared
 Figure 3. Tunable core-shell CdTe@CdS QDs with high
stability. Reproduced from Ref. 58. Figure 4. Preparation of sandwich-like SiO2@CdTe@SiO2
nanoparticles for cell labeling. Reproduced from Ref. 72.
(MSA)-capped CdTe/CdS QDs using stable Na2TeO3 as the Te
source via a one-pot reaction under neutral conditions (Figure dihydrolipoic acid (DHLA) are often used. Unfortunately, ligand-
3).58 A novelty of this approach is the use of MSA, which exchange can lead to a huge loss of fluorescence due to the
exhibits the features (pKCOOH1 = 3.30, and pKCOOH2 = 4.94) of changes in surface properties. These small molecules cannot
5 both TGA (pKCOOH = 3.53) and MPA (pKCOOH = 4.32) in terms of
45 prevent QDs from oxidation and degradation. Thereby a number
acidity. MSA can effectively stabilize QDs in a wider pH range of polymers (e.g. polyethylene glycol (PEG), and
(pH = 6 – 9) with better protection because of its stronger polyethylenimine (PEI)), proteins, peptides, and liposomes have
interactions with the surface Cd 2+ ions and its stronger steric been adopted to coat QDs.12 Similar to small-molecule modified
effects. In addition, slow decomposition of MSA-Cd complexes QDs, these surface-coated flexible polymers and biomolecules
10 forms a thin layer of CdS on the surface of CdTe nanocrystals,
50 are less resistant to oxygen and chemicals, and have little impact
decreasing the surface defects and leading to high fluorescence on the improvement of the photo- and chemical stability of QDs.
QY. Another novelty is the use of sodium citrate as buffer. The Therefore, organic-modified QDs still face the issues of toxicity,
resultant QDs show higher fluorescence QY than those stabilized instability and the loss of fluorescence, despite the significant
with TGA or MPA obtained from the conventional aqueous progress achieved in recent years.
15 method. They also show lower cytotoxicity at certain 55 Compared with unmodified and organically modified QDs,
concentrations due to the unique structure of MSA and the QDs coated with an inorganic shell show higher stability in terms
formation of a CdS shell on the surface of the CdTe core.58 of both chemistry and fluorescence. Silica is one of the most
In addition to organic and aqueous routes, QDs can be popular inert materials used for surface modification, and has a
produced in living organisms. Stürzenbaum et al. demonstrated few distinct advantages,65 including: (1) a non-porous silica shell
20 that the earthworm’s metal detoxification pathway can be
60 can protect QDs from environmental damage and improve their
exploited to produce water-soluble and biocompatible CdTe stability;66 (2) the silica shell can effectively inhibit the release of
QDs.59 This bioapproach is time-consuming (11 days), however, toxic Cd2+ ions and thus reduce the QDs’ toxicity;66 (3) the silica
and the resultant QDs have a low fluorescence QY (8%), so this coating can provide a hydrophilic surface and functional groups
method cannot be used for large-scale preparation. for conjugating with biomolecules.67 The silica shell can be
65 formed on the surface of the QDs by the Stöber method68 or the
25 2.2 Surface modification of QDs reverse microemulsion approach.69 Both methods have their own
From the viewpoint of bioapplications, QDs should have advantages and disadvantages, but one common challenge is the
excellent water-solubility, biocompatibility, and stability. These preparation of highly fluorescent QDs@SiO2 nanoparticles with
properties not only depend on their particle size, shape and tuneable size, as the fluorescence of QDs is drastically decreased
composition, but also rely on their surface structure and surface 70 during silica coating.
30 charge. More importantly, the surface properties of QDs In 2004, Nann et al. prepared single-dot@SiO2 nanoparticles
determine their bio-interface interactions, cellular endocytosis by the Stöber method.70 Yang and his co-workers prepared
and intracellular distribution, in vivo biodistributions, metabolism, similar CdTe@SiO2 nanoparticles by the reverse microemulsion
and fate.60-63 Engineering surface of QDs therefore becomes approach.69 These single-dot@SiO2 nanoparticles show a low
highly important as this process can improve these properties and 75 fluorescence QY (< 10%), however. Later on, the fluorescence
35 introduce additional functions.10 Medintz et al. recently QY of CdTe@SiO2 nanoparticles was improved to 47%. In
summarized the strategies for surface modification and comparison with incubated CdTe QDs (83%), nearly 40% of the
bioconjugation of QDs.11 One popular strategy for hydrophobic fluorescence was still lost during silica coating.71 The formation
QDs is ligand exchange, which not only transfers them from of single-dot@SiO2 nanoparticles is attributed to the electrostatic
organic solvents into aqueous solution, but also provides 80 repulsion between QDs and silica intermediates.
40 functional groups for further conjugation with biomolecules.64 In order to improve the fluorescence QY and the number of
Small water-soluble molecules such as TGA, MPA, and QDs in each SiO2 nanoparticle, we successfully prepared
sandwich-like SiO2@CdTe@SiO2 (SQS) nanoparticles using a
 Figure 6. (a) Structure of a multifunctional QD probe; (b)
Figure 5. (a) Size-dependence of SiO2@CdTe@SiO2 C4-2 cells labelled with multifunctional QDs; (c) In vivo
nanoparticles on tetraethyl orthosilicate (TEOS) volume; (b- targeted imaging using multifunctional QDs; (d) multicolour
d) size-dependence of cytotoxicity and cell uptake. capability of QD imaging in live mouse. Reproduced from
Reproduced from Ref. 74. Ref. 76.

novel strategy (Figure 4).72 We started from the synthesis of the QDs were internalized into the cells in the presence of transferrin
thiol-capped SiO2 core. The surface thiol groups can tightly 40 due to the occurrence of receptor-mediated endocytosis.
anchor CdTe QDs on the surface of SiO2 nanospheres. Then, a Motivated by the above pioneering research, extensive
silica layer was coated on the SiO2@CdTe to form SQS nonspecific and targeted bio-labelling and imaging have been
5 nanoparticles. During the silica coating, it is important to add an
carried out at different levels, ranging from in vitro to in vivo
appropriate amount of 3-mercaptopropyl-trimethoxysilane (MPS) models.10-12 Nonspecific cellular labelling involves the use of
for pre-coating in order to get highly fluorescent sandwich-like 45 hydrophobic and electrostatic interactions between surface-
nanoparticles. Compared with other QDs@SiO2 nanoparticles, capping molecules of QDs and biomolecules in the cell
our SQS nanopaticles show the highest fluorescence QY ever membrane. Thus, their surface ligand properties and the cell type
10 reported (up to 61%). They also show higher stability and lower largely determine the nonspecific adsorption and uptake of QDs.
toxicity in comparison with SiO2@CdTe nanoparticles. In most cases, such nonspecific adsorption is unwanted, as this
During the modification of QDs, the overall particle size has to 50 reduces the selectivity and targeting efficiency. In order to

be strictly controlled because it can dramatically influence the overcome nonspecific adsorption, PEG and its derivatives have
nanoparticle biological behaviour, such as cell internalization, been used to modify the QD surface, as they can effectively
15 tumour targeting and penetration, in vivo systemic and lymphatic minimize and prevent the nonspecific interactions of QDs with
biodistribution, metabolism, and clearance. Nanoparticles with a biomolecules, cells, and tissues.
size of 20-60 nm have shown distinct biodistribution, tumour 55 Similar to the in vitro nonspecific adsorption of cells, non-
penetration, and cellular tracking properties.73 Therefore, we targeted QDs can accumulate within tumours through the
prepared a series of SQS nanopaticles with sizes in the range of enhanced permeability and retention (EPR) effect. Such passive
20 39 nm to 76 nm by controlling the reaction parameters, including targeting is attributed to the leakiness of the tumour vasculature
the amount and the type of silica precursor, the ratio of silica and the poor lymphatic drainage, which enables QDs or other
75
precursor to ammonia, and the ratio of H2O to surfactant.74 These 60 nanoparticles to accumulate in tumours. The EPR effect could
SQS nanoparticles exhibited strong size dependence of their lead to more than 50 times as great nanoparticle accumulation in
stability, toxicity, and cellular uptake (Figure 5). Our findings tumours compared with healthy tissues. It is difficult, however, to
25 highlight the importance of controlling particle size and shell
maximize nanoparticle accumulation through the EPR effect, as
thickness during the preparation of fluorescent QDs@SiO2 core- this effect varies from tumour to tumour, and strongly depends on
75
65 the particle size and the surface charge. In addition, the EPR
shell nanoparticles.
effect is not commonly observed in some types of cancers such as
2.3 Fluorescence imaging of QDs gastric and pancreatic cancers.
The earliest bioapplications of fluorescent QDs were reported in An alternative approach is active targeting, which can be
30 1998.64, 67 Bruchez et al. coated core-shell CdSe@CdS QDs with achieved by conjugating QDs with targeting moieties such as
70 small molecules (e.g. folic acid and hyaluronic acid), peptides
a thin layer of silica and then conjugated them with biotin.67 The
biotinylated QDs were successfully applied to label 3T3 mouse (e.g. arginine-glycine-aspartic acid (RGD)), and proteins (e.g.
fibroblast cells. Chan et al. used small molecule TGA to transfer antibodies, antibody fragments, transferrin, etc.).12 In 2004, Gao
hydrophobic CdSe@ZnS QDs into water solution, and then and colleagues reported a landmark work on in vivo cancer
35 conjugated them with transferrin proteins.64 The authors targeting with QDs (Figure 6). 76 They first encapsulated
75 hydrophobic CdSe@ZnS core-shell QDs with an ABC triblock
incubated TGA-modified QDs and transferrin-QD conjugates
with HeLa cells, respectively, and found that no QDs could be copolymer (i.e. polybutylacrylate-polyethylacrylate-
observed inside the cell in the absence of transferrin. In contrast, polymethacrylic acid) by using hydrophobic-hydrophobic
 interactions between the capping ligands of QDs and the toxicity (Figure 5).74 Some research has shown that the release of
hydrophobic segments of the block copolymer. Then, they Cd2+ and the oxidation products of anions are responsible for
66
conjugated tumour-targeting ligands and drug-delivery 60 their bio-toxicity. The QDs themselves (i.e. non-degraded QDs)
functionalities with the polymethacrylic acid segment. The in are not acutely toxic, and they can be retained in the body for two
5 vivo study showed that these QD probes accumulated at the years and remain fluorescent.
tumour site through the EPR effect, and the specific antibody- In 2007, Choi and co-workers studied the renal clearance of
antigen interactions. It is worth mentioning that passive targeting QDs.83 They chose cationic, anionic, zwitterionic, and neutral
is much slower and less efficient than active targeting. 65 molecules to modify CdSe@ZnS core-shell QDs and tested their

Although targeted nanoparticles hold much promise, and the binding with serum proteins. They found that the QD surface
10 concept was introduced more than 30 years ago, none of them has charge has a profound effect on the adsorption of serum proteins
been clinically approved.75 One possible reason is the huge gap and the hydrodynamic diameter. Cationic or anionic charge led to
between cost and benefit. Compared with expensive antibodies the hydrodynamic size increasing from around 5 nm to over 15
and other targeting ligands, cost-effective small molecules such 70 nm after incubation with serum. Neutral (PEGylated) QDs did not

as folic acid have been adopted. Folic acid and folate conjugates aggregate, but had a large size. Zwitterionic coatings prevented
15 can be specifically recognized by the folate receptor (FR), which serum protein adsorption and produced the smallest
is a glycosylphosphatidylinositol-anchored protein. The alpha hydrodynamic size. The biodistribution results show that a final
isoform of FR (FR-α) is found to be overexpressed in many hydrodynamic diameter < 5.5 nm resulted in rapid and efficient
epithelial cancers, but not highly expressed in normal tissues 75 urinary excretion and elimination of QDs. In their later report, the

except for the kidney. Since the affinity of FR to folic acid and authors conjugated small targeting molecules on the surface of
20 folate conjugates is relatively high (Kd ≈ 100 pM), FR-α has been zwitterionic coatings of QDs.84 These targeted probes were also
extensively investigated for tumour targeting,77 including many cleared by the kidneys when their hydrodynamic size was smaller
studies focusing on QDs. For example, folic acid was conjugated than 5.5 nm, which sets an upper limit of 5–10 ligands per QD for
to PEG and subsequently deposited onto N-acetyl-L-cysteine 80 renal clearance. The animal models demonstrated their
(NAC)-stabilised CdTeS QDs, which was demonstrated to be performance for in vivo targeted imaging and renal clearance
78
25 able to target tumours in mouse models. Another small targeting within 4 h post-injection.
molecule is hyaluronic acid, which is widely distributed Recently, Ye et al. injected phospholipid micelle-encapsulated
throughout connective, epithelial, and neural tissues. Hyaluronic CdSe/CdS/ZnS QDs into rhesus macaques, and tracked the
85
acid, associated with tumour angiogenesis and progression,79 has 85 relevant markers in the next 90 days. Their results demonstrated
been conjugated to QDs for tumour targeting, as it can that the acute toxicity of these QDs in vivo is minimal.
30 specifically bind with CD44, a cell-surface glycoprotein Accumulation of an initial dose of Cd was found in the liver,
overexpressed in many tumour types. Therefore their conjugates spleen, and kidneys, however, even after 90 days, indicating slow
have not only cancer targeting characteristics, but also the breakdown and clearance of the QDs. Although QDs have not
capability for imaging lymphatic vessels. 80 90 shown acute or short-term toxicity, comprehensive assessments

In addition to the high cost, the low targeting efficacy and the of their long-term bio-toxicity are needed to confirm the ultimate
35 unclear mechanism could also limit their clinical applications. fate of these heavy metals and the impact of their persistence in
This is because not all cancer cell types overexpress the same primates for potential clinical use.
unique receptors, and the overexpressed receptors are often
present on normal cells.75 Moreover, the density of the targeted
95 3 Fluorescent metal nanoclusters
receptors on tumour cells could be another factor influencing the
40 targeting efficacy. For II-VI QDs, the biggest challenge for their Since QDs have potential toxicity and long in vivo retention time,
clinical applications is their potential toxicity, as discussed in the many efforts have been made to develop alternatives to them. An
following section. alternative is fluorescent metal nanoclusters, which have attracted
considerable attention during the past several years. It is well
2.4 QD toxicity
100 known that large nanoparticles of metals such as Au, Ag, and Cu
Most II-VI QDs consist of toxic elements such as cadmium, possess the face-centred cubic (fcc) structure and the surface
45 lead, mercury, etc. Their toxicity has always been of concern and plasmon resonance (SPR) property.86 Their SPR absorption is due
could limit the diversity of their applications, such as in solar to the collective oscillation of electrons on the surfaces, and it is
cells, light-emitting diodes, flat-screen televisions, and strongly dependent on the particle size. When that size is smaller
biomarkers.81 The bio-toxicity depends on multiple factors,82 105 than the electron mean path (e.g. 20 nm for Au nanoparticles), the
which can be mainly classified into two groups: (1) the inherent conducting electrons in the ground states and excited states are
50 properties of QDs, including QD size, charge, composition, confined.86 The large metal nanoparticles have very low
concentration, and outer-layer coating bioactivity (capping fluorescence emission. Very interestingly, when their size is
material, functional groups); (2) environmental factors such as further reduced below 2 nm, the ultra-small nanoclusters possess
oxidation, photolysis, and mechanical effects. A number of 110 different crystal structures and exhibit strong photoluminescence
studies show that appropriate surface modification, modulating while their unique SPR property disappears.
55 the surface charge, and controlling the QD dosage can effectively Nanoclusters bridge the gap between molecules and
reduce QD cytotoxicity. Previously, we demonstrated that coating nanoparticles, and could simultaneously display the properties of
QDs with silica shells can improve their stability and reduce the both molecules and nanoparticles. Their novel optical, electronic,
 and catalytic activities make them very useful in ultrasensitive
detection, biolabelling, bioimaging, and catalysis.87-90 The big
challenge, however, is how to controllably synthesize metal
nanoclusters with defined size, composition, crystal structure, and
5 surface properties.88, 91
3.1 Synthesis of fluorescent metal nanoclusters
Compared with large nanoparticles, metal nanoclusters are
difficult to synthesize and functionalize because they only consist
of a few to tens of metal atoms. They are very sensitive to slight
10 variation of the environment, such as solution pH, ion strength,

solvents, oxygen, temperature etc. They have very high surface-

area-to-volume ratios and tend to aggregate into large particles. In
general, fluorescent metal nanoclusters can be prepared by
reduction of metal precursors or etching of large nanoparticles in
15 the presence of strong stabilizers such as small thiol-molecules,

polymers, and biomolecules.

Reduction of metal precursors such as salts and complexes is a
straightforward way to produce fluorescent metal nanoclusters.
Au nanoclusters are usually chosen as representative for
20 investigation due to their high chemical stability, easy
preparation, and biocompatibility. The first observation of Au
photoluminescence from its ingots, single-crystal slices, and
films, with a QY of 10-10, was reported by Mooradian in 1969.92
The extremely low QY did not attract any attention until
25 Wilcoxon et al. observed fluorescence from colloidal Au
nanoparticles with a QY of 10 -5 – 10-4.93 The authors prepared
colloidal Au nanoparticles through reduction of HAuCl4 by
citrate in water, or by metallic sodium dispersion or lithium
trisamylborohydride in inverse micelles, and then used liquid
30 chromatography to fractionate the resultant Au nanoparticles.

They found that only nanoparticles smaller than 5.0 nm showed a

blue fluorescence at 440 nm under an excitation of 230 nm. Their
results suggest that ultra-small nanoclusters could exhibit strong
fluorescence.
Figure 7. Schematic diagram of preparation of (a) polymer
35 A breakthrough in preparing fluorescent Au nanoclusters was
ligand PTMP-PMAA; (b) photoreductive synthesis of
achieved by Zheng and co-workers.94-96 They synthesized a series fluorescent Cu, Ag, and Au nanoclusters; (c) TEM image of
of Au5, Au8, Au13, Au23, and Au31 nanoclusters using Au nanoclusters. Reproduced from Ref. 100 and Ref. 105.
poly(amidoamine) (PAMAM) dendrimers as stabilizers. By
adjusting the molar ratio between Au3+ and PAMAM from 1:1 to polymer ligands did not show fluorescence, and the observed
40 1:15, they tuned the emission of these Au nanoclusters from the
fluorescence was only caused by the Au nanoclusters. The
ultraviolet (UV) to the near infrared (NIR) range with a QY from 60 different emissions of Au nanoclusters are attributed to their
10% to 70%. The latter experiments, however, proved that different sizes. On the basis of this research, we prepared
PAMAM dendrimers made a contribution to the solution fluorescent Au, Ag, and Cu nanoclusters using photoreduction
fluorescence. The authors also used dendrimers as ligands to rather than chemical reduction (Figure 7(b)).105 Compared with
45 prepare fluorescent Ag nanoclusters.89, 97 In addition to conventional chemical reduction, photoreduction is clean and
dendrimers, some other polymers such as multiarm star 65 non-toxic as this method avoids the use of additional reducing
polyglycerol-block-poly(acrylic acid) and DHLA functionalized agents. The QYs of the resultant Au, Ag, and Cu nanoclusters
PEG were used to stabilize metal nanoclusters. 98, 99 were 5.3%, 6.8%, and 2.2%, respectively. Using the
Recently, we used multidentate thioether-terminated photoreduction method, Shang et al. also prepared very highly
50 poly(methacrylic acid) (PTMP-PMAA) (Figure 7(a)) as ligand to
fluorescent Ag nanoclusters (18.6% QY) in the presence of
successfully prepare water-soluble fluorescent Au nanoclusters 106
70 poly(methacrylic acid) (PMAA).
through reduction of HAuCl4 with NaBH4.100 Due to the strong Compared with PMAA, our polymer ligands have a stronger
steric effect, this polymer ligand has also been used to prepare steric hindrance effect. Figure 7(c) presents a typical transmission
ultra-small magnetic iron oxide nanoparticles.101-104 By electron microscope (TEM) image of Au nanoclusters stabilized
55 controlling the polymer concentration and molecular weight, we
with PTMP-PMAA, clearly showing their ultra-small size (< 1.0
obtained a series of Au nanoclusters with emissions between 540 75 nm). In order to further investigate the polymer hindrance effect,
– 800 nm and QYs of 2.6 – 4.8%. In contrast to dendrimers, our we designed three types of tridentate thioether-terminated
 polymer ligands,107 i.e. poly(methyl methacrylate) (PTMP-
PMMA), poly(n-butyl methacrylate) (PTMP-PBMA), and poly(t-
butyl methacrylate) (PTMP-PtBMA), which were used to
synthesize fluorescent Au nanoclusters through the facile
5 photoreduction method. The resultant Au nanoclusters exhibited

blue fluorescence instead of red fluorescence due to their small

particle size. Their QYs were found to be 3.8%, 14.3%, and
20.1%, respectively, which increases with increasing polymer
steric hindrance, i.e. PTMP-PMMA < PTMP-PBMA < PTMP-
10 PtBMA.

In addition to polymer ligands, small thiol molecules such as

glutathione, tiopronin, MPA, DHLA, phenylethylthiolate, and
thiolate α-cyclodextrin were also used to prepare fluorescent
metal nanoclusters.87, 108, 109 For example, Luo et al. used L-
15 glutathione as ligands to prepare Au(0)@Au(I)-thiolate core-shell

nanoclusters with a QY of 15%.109 They proposed that strong

luminescence emission is attributed to aggregation-induced
emission of Au(I)-thiolate complexes. The QYs of metal
nanoclusters stabilized by small molecules are similar to those Figure 8. (A-B) Solutions of Ag7,8 and alloyed Ag7Au6
20 nanoclusters stabilized with polymer. nanoclusters; (C-D) their absorption and emission spectra;
In order to improve the biocompatibility of fluorescent metal (E) alloyed Ag7Au6 nanoclusters in solution and in the solid
nanoclusters, several groups used biomolecules such as state under visible and UV light; (F) comparison of the
oligonucleotides, peptides, and proteins as stabilizers during PAGE of Ag7,8 and alloyed Ag7Au6 nanoclusters.
preparation.110-113 For example, the Dickson group took Reproduced from Ref. 117.
+
25 advantage of the strong affinity of Ag to cytosine bases from stabilized Ag nanoparticles and then added them into an organic
single-stranded DNA, and prepared very small Ag nanoclusters 60solvent (e.g. toluene, carbon tetrachloride, diethyl ether)
using DNA as stabilizer.110 In their later report, they used DNA containing excess MSA under magnetic stirring. A mixture of
microarrays for high-throughput analysis of 12-mer strands to Ag8 and Ag7 nanoclusters with red and blue-green fluorescence
identify optimized sequences for Ag encapsulation, and produced was obtained. The QYs of the Ag8 nanoclusters at room
111
30 five distinct Ag emitters with QYs in the range of 16 – 34%. temperature and 273 K were calculated to be 0.3% and 9%,
Compared with single-stranded DNA, proteins have abundant 65 respectively. The authors used a similar approach to obtain

binding sites and offer better protection to metal nanoclusters. alloyed Ag7Au6 nanoclusters (3.5% QY) by adding HAuCl4
Xie et al. prepared Au25 nanoclusters with a QY of 6.0% using solution into the as-etched Ag nanocluster solution (Figure 8).117
bovine serum albumin (BSA) as the stabilizer and reducing In addition to small molecules, multivalent polymers can also
112
35 agent. The reduction process was induced by adjusting the be used as etching agents. Duan et al. used multivalent
solution pH. 70 polyethylenimine (PEI) to etch 8 nm Au nanoparticles, which

Similar to the QDs produced in living organisms, fluorescent were prepared by a two-phase approach and stabilized with
metal nanoclusters can also be formed in-situ in cells. For dodecylamine. The resultant cluster solution surprisingly
example, Wang and co-workers found that fluorescent Au appeared to be in an oxidized electronic state with an emission at
40 nanoclusters were spontaneously biosynthesized by cancer cells 505 nm. The emission was blue shifted to 445 nm with a QY of
(human hepatocarcinoma cell line HepG2 and leukaemia cell line 118
75 10 - 20% after reduction with NaBH4.

562) rather than normal cells such as human embryo liver cells Similar to organic ligands, metal precursors can also induce
(L02) when the cells were incubated with chloroauric acid the etching process. For example, Lin and co-workers extracted
solution.114 Au nanoclusters were formed by reduction of Au- HAuCl4 from aqueous solution into
45 precursor inside the cell cytoplasm and concentrated around their didodecyldimethylammonium bromide (DDAB) toluene solution,
nucleoli. The selective formation of fluorescent Au nanoclusters 80 and then added the mixture into 5.6 nm Au solution to result in

by cancer cells can be exploited for in vivo self-bio-imaging of 3.2 nm particles.119 After replaced DDAB with dihydrolipoic
tumours. acid, these Au nanoparticles were further decreased to 1.6 nm and
showed a red emission around 700 nm. Their fluorescence QY
Etching of large metal nanoparticles is an alternative approach to
was 3.4% in methanol and 1.8% in water (pH = 9). Recently,
50 prepare fluorescent metal nanoclusters. The etching process can
85 Yuan et al. developed a general etching approach to prepare
be performed by adding strong ligands or precursors into the
fluorescent Au, Ag, Cu and Pt nanoclusters with a QY of 5.4%,
nanoparticle solution. For example, Muhammed et al. synthesized
6.5%, 3.5% and 4.6%, respectively. 120 They started with
fluorescent Au nanoclusters from MSA-stabilized Au
glutathione-stabilized metal nanoparticles, and then transferred
nanoparticles by etching with excess glutathione.115 The etching
the metal nanoparticles into an organic phase by taking advantage
55 process is pH-dependent and the obtained Au 8 and Au25
90 of the electrostatic interactions between negatively charged
nanoclusters have a QY of 0.015% and 0.19%, respectively. They
glutathione (carboxyl group) and positively charged
also developed an interfacial etching process to prepare
116 cetyltrimethylammonium bromide (CTAB). The beauty of this
fluorescent Ag nanoclusters. First, they prepared MSA-
 approach is that the resultant fluorescent metal nanoclusters can
be shuttled back to the aqueous phase using hydrophobic-
hydrophobic interactions upon addition of hydrophobic salts (e.g.
tetramethylammonium decanoate) in chloroform.
5 Besides these methods, microwaves and ultrasound were also
used to assist the synthesis of fluorescent metal nanoclusters in
recent years.121, 122 For example, Xu and Suslick adopted
sonochemistry to prepare fluorescent Ag nanoclusters with a QY
of 11% in the presence of PMAA.121 Shang et al. synthesized
10 fluorescent Au nanoclusters (2.9% QY) via a rapid microwave
assisted method.122 In all syntheses, ligands play a crucial role in
obtaining these ultra-small fluorescent metal nanoclusters. Their
ability to donate electrons drastically influences the fluorescence
intensity, i.e. the stronger the electron donating capability is, the
15 higher the fluorescence intensity will be. 123
3.2 Characterization and modification of fluorescent metal
nanoclusters Figure 9. (a) Kohn-Sham orbital energy level diagram for a model
compound Au25(SH)18; (b) Solid-state model for the origin of the
Metal nanoclusters can be characterized by the techniques applied
two luminescence bands in (d); (c) theoretical absorption spectrum
to nanomaterials and molecules. Compared with large of Au 25(SH) 18; (d) two luminescence peaks observed in Au 28(SG) 16
20 nanoparticles, metal nanoclusters have a smaller size and a clusters. Reproduced from Ref. 131 and Ref. 132.
“narrower” size distribution, so that size-selective precipitation is
The ultra-small size (limited atomic numbers) of metal
not suitable for their separation. They are usually fractionated by
nanoclusters makes it possible to predict their crystal structures
chromatography and electrophoresis techniques, which are
through precise theoretical simulation. For example, Xiang et al.
usually applied to molecules. These separation methods include
60 developed a new genetic algorithm approach to search for the
25 high-performance liquid chromatography (HPLC), size exclusion
global lowest-energy structures of DMSA-stabilized Ag
chromatography (SEC), ion exchange chromatography (IEC),
nanoclusters.129 In combination with density functional theory
capillary electrophoresis, and polyacrylamide gel electrophoresis
(DFT), their genetic algorithm simulations show that the ground
(PAGE). It is still very challenging to obtain monodisperse
state of [Ag7(DMSA)4]− has eight instead of four Ag−S bonds,
nanoclusters using these approaches. For example, Tsunoyama et
65 with a much lower energy than the structure based on the
30 al. separated Au:SCx nanoclusters into different fractions using
[Ag7 (SR)4 ]− cluster with a quasi-two-dimensional Ag7 core. Their
gel permeation chromatography (GPC),124 and then characterized
simulated X-ray diffraction pattern of the [Ag7(DMSA)4]− cluster
them with laser-desorption ionization (LDI) mass spectroscopy.
is in good agreement with the experimental results.
The results show that each fraction had a wide distribution of Au
The optical properties of fluorescent metal nanoclusters can be
atoms although they were well separated with high resolution in
70 characterized with UV-visible (UV-Vis) absorption and
35 the GPC spectrum. Negishi and co-workers synthesized
photoluminescence spectroscopy. As mentioned previously,
glutathione-protected Au nanoclusters and then fractionated them
metal nanoclusters have no SPR absorption, but they show
into 9 fractions, with the number of Au atoms ranging from 10 to
molecular-like electronic transitions due to the quasi-continuous
39 by PAGE analysis.125 Among their Au nanoclusters,
energy band structure and quantum confinement effects. Bakr et
Au25(SG)18 is the most stable one.
75 al. synthesized Ag nanoclusters through the reduction of Ag-
40 The size of fractionated metal clusters can be characterized by
precursor in the presence of 4-fluorothiophenol,130 and
TEM, and their molecular weight can be measured by mass
investigated the evolution of their absorption from multiple bands
spectroscopy. In principle, their crystal structures could be
into a single SPR band by heating the original nanocluster
determined by X-ray diffraction (XRD). Metal nanoclusters are
solution at 90 ˚C for different periods of time. Their results
less ordered, however, and their powder XRD patterns are broad.
80 demonstrate the size dependence in UV-Vis absorptions of metal
45 In comparison with metal complexes with defined molecular
nanoclusters.
structure, it is very difficult to obtain single crystal clusters for
In order to demonstrate the origin of multiband absorption,
structural characterization. So far, most structural investigations
Zhu and co-workers chose the Au25(SR)18 cluster as a model and
of metal nanoclusters are focused on “large” Au nanoclusters.91,
108, 126-128 simulated their absorption by performing time-dependent DFT
For example, Jadzinsky et al. determined the structure 131
85 calculations. Figure 9(a) shows the Kohn-Sham molecular
50 of a Au 102(p-mercaptobenzoic acid)44 single crystal and found a
orbitals, energies, and atomic orbital contributions in the cluster.
core-shell structure,127 in the which the Au49 core is surrounded
The highest occupied molecular orbital (HOMO) and the lowest
by two groups of Au atoms. Qian and co-workers characterized
three lowest unoccupied molecular orbitals (LUMOs) are mainly
the crystal structure of Au25(SR)18 and Au38(SR)24 nanoclusters,
composed of 6sp atomic orbitals of Au, and these orbitals
and found a similar core-shell structure.91 An Au25(SR)18 cluster
90 constitute the sp-band. The HOMO-1 to HOMO-5 orbitals are
55 consists of an icosahedral Au 13 core and exterior 12 Au atoms in
constructed from the 5d atomic orbitals of Au and form the d-
the form of six –RS–Au–RS–Au–RS– motifs.91, 108
band. In addition, the s 3p orbitals make contributions to both sets
of HOMO and LUMO orbitals. The multiband absorption of
 metal nanoclusters suggests their multiple emission peaks and
broad fluorescence spectra (Figure 9(b)). Figure 9(c) shows the
simulated absorption spectrum. The multiple absorptions are
attributed to the intraband (sp) HOMO → LUMO transition, the
5 interband transition (d → sp), or mixed sp → sp intraband and d

→ sp interband transitions.131 Figure 9(d) shows two fluorescence

bands with the maxima at around 1.5 and 1.15 eV observed in
Au28(GSH)16 nanoclusters by Link et al.132 These two bands are
separated from a broad luminescence in the range of 2.0 – 0.8 eV,
10 and are ascribed to the radiative interband recombination between

the sp and d bands, and intraband transitions (sp bands) between

the HOMO and LUMO.
Despite the good agreement between simulated data and
experimental observations, the origin of metal fluorescence is not
15 completely understood. Most reported atomically precise
Au n(SR)m nanoclusters show very weak luminescence. Recently,
Yu and co-workers identified that Au22(SR)18 has two RS-[Au-
SR]3 and two RS-[Au-SR]4 motifs that are interlocked and
capped on a prolate Au8 core.133 These Au22(SR)18 nanoclusters
20 exhibited an emission at ∼665 nm with a QY of ∼8%. Their

results show that the luminescence of these core-shell

nanoclusters was generated by the aggregation-induced emission
of Au(I)-thiolate complexes on the nanocluster surface.
The fluorescence of metal nanoclusters is very sensitive to the
25 cluster size, surface ligands, solvents, etc., so it is thus necessary

to modify them in order to maintain their bright fluorescence in

addition to improving their stability and biocompatibility. There
are few reports, however, on the post-modification of fluorescent
metal nanoclusters in comparison with large nanoparticles, due to
30 their tiny size and sensitivity to external conditions. Lin and co-
Figure 10. Comparison of cell labeling by using fluorescent
workers prepared DHLA-protected fluorescent Au nanoclusters Au nanoclusters and CdTe QDs. Reproduced from Ref. 100.
by etching large nanoparticles and replacing surface ligands.119
labelling suspended and adherent hematopoietic relatively normal
They took advantage of carboxylic acid groups from DHLA to
cord blood mononuclear (CBMC) cells and cancer K562 cells
conjugate PEG–NH2 or biotin-PEG–NH2 with Au nanoclusters. 100
60 (Figure 10). The results show that the cancer cells took up
35 The gel electrophoresis and the cell labelling indicate the
more Au nanoclusters than the normal cells, even though they
successful conjugation. Samanta et al. prepared fluorescent Au
nanoclusters using a novel quaternary ammonium as the ligand, were from the same hematopoietic system. There was no
difference, however, in the uptake of CdTe QDs between the two
and then coated them with silica.134 Similar to fluorescent QDs,
kinds of cells. The selective uptake of Au nanoclusters by cancer
surface modification can lead to the fluorescence quenching of
65 cells could be attributed to the unique properties of Au
40 metal nanoclusters. It is still a great challenge to obtain robust
nanoclusters or the nature of the cells. In addition, CdTe QDs
fluorescent metal nanoclusters through surface modification.
destroyed the nuclei of some cells. We also compared the
3.3 Application of fluorescent metal nanoclusters in bioimaging cytotoxicity of Au nanoclusters with that of CdTe QDs through
MTT and apoptosis assay. The results show that our fluorescent
Similar to other fluorophores, fluorescent metal nanoclusters have
70 Au nanoclusters had lower toxicity than QDs, and did not induce
also been tested for in vitro and in vivo bioimaging. In the early
acute toxicity. These advantages make them very attractive in
45 reports, Zheng et al. prepared fluorescent Au, Ag nanoclusters in
selective bio-labelling of cancer cells. Retnakumari et al.
the presence of dendrimers, DNA, and proteins, and used them as
conjugated folic acid with BSA-stabilized Au nanoclusters and
labels for cell imaging.89, 94-97 Baskov et al. prepared fluorescent
then used them for targeted imaging. 136 The receptor-targeted
Ag nanoclusters in the presence of thioflavin T with remarkable +ve
75 cancer detection was demonstrated on FR oral squamous cell
fluorescent properties,135 and then used them to label amyloid
carcinoma (KB) and breast adenocarcinoma MCF-7 cells, where
50 fibrils produced from recombinant mammalian prion proteins and
the FA-conjugated Au25 clusters were found to be internalized in
non-prion proteins. The labelled amyloid fibrils exhibited a time-
significantly higher concentrations compared to the negative
dependent increase in fluorescence with no photobleaching after
control cell lines.136 Apart from routine utilization of cell
24-h illumination, while those stained with thioflavin T showed a
80 labelling, fluorescent metal nanoclusters can be used as
rapid decay in fluorescence. Their results demonstrate the higher
intracellular sensors. For example, Shang and co-workers
55 stability of Ag nanoclusters than that of organic fluorophore.
demonstrated the use of Au nanoclusters for intracelluar
Recently, we prepared fluorescent Au nanoclusters stabilized
thermometry by taking advantage of the temperature sensitivity
with PTMP-PMAA, and then compared them with CdTe QDs in
of their fluorescence lifetime and emission intensity (Figure
 Figure 12. (a) Biodistibution of 2-nm GS-Au nanoclusters.
The inset shows CT images of a live mouse before and 30
min after injection of Au nanoclusters; (b-d) comparison of
biodistribution of GS-Au nanoclusters and IRDye 800CW.
Figure 11. FLIM images of HeLa cells with internalized Au Reproduced from Ref. 138 and Ref. 139.
nanoclusters at four different temperatures. Reproduced from
Ref. 15. that an injection of insulin-Au nanoclusters into the rats tended to
reduce the blood glucose in a similar way to commercial insulin.
11).15 Using fluorescence lifetime imaging microscopy (FLIM), Fluorescent insulin-Au nanoclusters can also be used as contrast
they observed the considerable variation of fluorescence lifetime agents for CT imaging.113 These studies indicate that ultra-small
of nanoclusters internalized in HeLa cells with the temperature 45 fluorescent Au nanoclusters could simultaneously serve as very
increasing from 15 to 45 ˚C. promising contrast agents for in vivo fluorescence imaging and
5 In addition to the above in vitro cell labelling and imaging, Wu CT imaging.
et al. investigated in vivo imaging through the tail vein In summary, fluorescent metal nanoclusters as emerging
administration of near infrared (NIR) fluorescent Au nanoclusters fluorophores have attracted considerable attention due to their
in live mice,137 and found that the uptake of NIR Au nanoclusters 50 tuneable emissions, ultra-small size, fast renal clearance, and low
by the reticuloendothelial system was relatively low in toxicity. There are a few obstacles to be overcome, however,
10 comparison with other nanoparticles due to their ultra-small including (i) low fluorescence QY, which is usually about ~10%
hydrodynamic size (~2.7 nm). They then used MDA-MB-453 and and less than that of QDs and many organic dyes; (ii)
xenografted HeLa tumour cells as models to do in vivo and ex polydispersity in size and components, which makes it very
vivo imaging studies, and found that the ultra-small Au 55 difficult to fundamentally study their novel properties and
nanoclusters were highly accumulated in the tumour areas due to mechanisms; (iii) difficulty in modifying their surface to
15 the enhanced permeability and retention (EPR) effect. 137 introduce other functions due to their tiny size and lower stability;
Zhou and co-workers studied the renal clearance of 2 nm (iv) complicated interactions with biological environments.
glutathione (GSH)-coated fluorescent Au nanoclusters (Figure
12),138 and found that only ~4% of the particles were accumulated
in the liver, while more than 50% of the particles were found in 60 4 Fluorescent carbon nanomaterials
20 urine within 24 h after intravenous injection, which is comparable 4.1 Fluorescent carbon dots
to the QDs with the best renal clearance efficiency.83 They also
used computed tomography (CT) to visualize real time Fluorescent carbon dots are also used as alternatives to QDs for
accumulation of luminescent GS-AuNPs in the bladder, and bioimaging,17, 140 because they not only exhibit several favourable
demonstrated that fluorescent Au nanoclusters can serve as attributes of traditional semiconductor-based QDs (namely, size-
25 contrast agents for CT imaging (Figure 12). Recently, they 65 and wavelength-dependent emission, resistance to
compared GSH-coated fluorescent Au nanoclusters (2.5 nm) with photobleaching, ease of bioconjugation), but also show chemical
small dye molecules IRDye 800CW,139 and found that they both inertness, low toxicity, and biocompatibility. Fluorescent carbon
have similar physiological stability and renal clearance, but Au dots were accidently discovered in 2004 during the purification of
nanoclusters exhibited a much longer tumour retention time and single-wall carbon nanotubes (SWCNTs) fabricated by the arc-
141
30 faster normal tissue clearance (Figure 12). These merits enabled 70 discharge approach. Two new classes of nanomaterials were
the Au nanoclusters to detect the tumour more rapidly than the isolated from the crude soot. One was short, tubular carbon, and
dye molecules without severe accumulation in reticuloendothelial the other a mixture of fluorescent nanoparticles derived from the
system organs.139 SWCNTs.
Besides the above in vivo passive targeting, fluorescent metal In 2006, Sun et al. obtained 5-nm non-fluorescent carbon dots
35 nanoclusters can be tagged with bioactive molecules for targeting, 75 via laser ablation of a carbon target, and then modified them with

imaging, and therapy. For example, Liu et al. synthesized PEG to get fluorescent carbon dots with a fluorescence QY of 4%
fluorescent Au nanoclusters (0.92 ± 0.03 nm) using insulin as a – 10%.142 The photoluminescence of these carbon dots was broad
template.113 The resulting Au-insulin nanoclusters retain the and strongly dependent on the excitation wavelength, which
insulin bioactivity and biocompatibility, and have been used to could be attributed to the different sizes in the sample and
40 regulate the in vivo glucose level in Wistar rats. The results show 80 different emission sites on the passivated particle surfaces. After

fractionation with gel column chromatography, most of the

 Figure 14. (a) NIR images of mouse bladders acquired
before and after injection of carbon dots through intravenous
injection, subcutaneous injection, and intramuscular
injection; (b) quantification of the ZW800 fluorescence
signal in (a); (c) representative coronal images from 1 h
dynamic positron emission tomography (PET) imaging.
Reproduced from Ref. 152.
exhibited bright fluorescence in both cell membrane and
cytoplasm regions under an excitation of 800-nm laser pulses.
The results demonstrate that carbon dots exhibit strong
35 luminescence with two-photon excitation in the near-infrared, and

moreover, large two-photon absorption cross-sections,

Figure 13. Digital images of solid fluorescent carbon dots, comparable to those of available high-performance
aqueous solutions, and their absorption, excitation and semiconductor QDs.150 The authors further demonstrated the in
emission spectra. Reproduced from Ref. 144.
vivo imaging of fluorescent carbon dots.151 They compared the
fluorescent fractions could achieve emission yields close to 40 imaging capability of carbon dots and ZnS-doped carbon dots,

60 %.143 Interestingly, their optical properties resemble band-gap and found that the later dots emitted more strongly than the
transitions, which are found in nanoscale semiconductors, former dots both in solution and in mice. The fluorescence from
suggesting that carbon dots have essentially semiconductor-like the bladder area was observed, and 3 h after injection, the
5 characteristics. Recently Bhunia et al. prepared hydrophobic and fluorescence could be detected in the urine, but it completely
hydrophilic carbon dots with tuneable size and visible 45 faded 24 h after injection. They analysed the biodistribution of

emissions,144 by dehydrating carbohydrate in octadecene in the carbon dots and found that the carbon dots accumulated in the
presence of octadecylamine, or in concentrated sulphuric acid kidney and, to a small extent, in the liver.151 This is attributed to
(Figure 13). Their method produced gram-scale fluorescent the surface PEG, which likely reduces the protein adsorption.
10 carbon dots with a QY of 6 – 30%. Zhu and co-workers also Recently Huang and co-workers investigated the effects of
reported a rapid and high-output hydrothermal approach to 50 injection routes on the biodistribution, clearance, and tumour

prepare polymer-like carbon dots with QYs as high as 80 %.145 uptake of carbon dots (Figure 14).152 They prepared fluorescent
In addition to solid fluorescent carbon dots, there are some carbon dots through a laser ablation approach, and then
reports on hollow fluorescent carbon dots.146, 147 For example, functionalized carbon dots with the NIR dye ZW-800 and the
15 Fang et al. simply mixed acetic acid, water and diphosphorus isotope 64Cu. They injected the conjugates into mice in three
pentoxide to obtain cross-linked hollow fluorescent carbon 55 different manners, i.e. intravenous, intramuscular, and
nanoparticles. By reducing the release of heat, they also obtained subcutaneous injection. The results show that the carbon dots
solid fluorescent nanoparticles. So far, many approaches, such as were efficiently and rapidly excreted from body after injection,
arc-discharge, laser ablation, electrochemical oxidation, and the clearance rate of carbon dots decreased when the
20 combustion/pyrolysis, and hydrothermal and microwave administration was varied from intravenous, to intramuscular, and
methods, have been developed to prepare solid and hollow 60 then to subcutaneous injection (Figure 14). Different injection

fluorescent carbon dots.148 The preparation is inexpensive on a routes also showed different blood clearance patterns and
large scale without the need for stringent, intricate, tedious, different tumour uptake of carbon dots.
costly, or inefficient steps.149 The recent advances in the synthesis
4.2 Fluorescent graphene quantum dots
25 and characterization of fluorescent carbon dots have been

reviewed.17, 148, 149 It should be noted that fluorescent graphene quantum dots
The first study of fluorescent carbon dots in bioimaging was 65 (GQDs), the analogues of carbon dots, have also attracted
reported by the Sun group in 2007.150 The authors used poly- considerable attention.153, 154 Similar to carbon dots, GQDs can be
(propionylethylenimine-co-ethylenimine) (PPEI-EI, with EI prepared by top-down and bottom-up approaches, and their
30 fraction ~20%) to modify the carbon dots, and then applied them fluorescence can be enhanced via surface modification. The top-
to label human breast cancer MCF-7 cells. These labelled cells down methods usually refer to cutting larger size carbon
 Figure 16. Nitrogen-doped GQDs for cellular and deep-
tissue imaging. Reproduced from Ref. 164.
30 was changed into blue after the GQDs were modified with
alkylamines or reduced with NaBH4 (referred to as m-GQDs and
r-GQDs respectively), while the particle size was similar. The
fluorescence shift was attributed to the suppression of non-
radiative processes and to the enhanced integrity of the π
35 conjugated system. These three types of GQDs exhibited strong

excitation-dependent down-conversion and up-conversion

emissions, demonstrated by that of the m-GQDs in Figure 15(e-
f),155 which is in contrast to Zhuo’s report [Figure 15(c-d)]. In
Figure 15. (a) Image of GQD solution under UV-light; (b) addition to the preparation, the optical properties of GQDs are
schematic emissions in GQDs; (c-d) excitation-independent 40 also influenced by the solution pH, solvent, and concentration.
154
down-conversion and up-conversion spectra of GQDs; (e-f)
Recently, Xu et al. studied the fluorescence of GQDs on a
excitation-dependent down-conversion and up-conversion
spectra of GQDs. Reproduced from Refs. 155, 157, 160, and substrate at the single particle level.160 All the GQDs investigated
161. had the same spectral lineshapes and peak positions, despite
notable differences in particle size and the number of layers.
materials such as carbon nanotubes, graphene or graphene oxide 45 GQDs with more layers were brighter than those with fewer

sheets, and carbon fibres into small GQDs, through strong acid layers, but were associated with shorter fluorescence lifetimes.
oxidation, hydrothermal or solvothermal treatment, or microwave Although there are some debates on the fluorescence
and sonication treatment.154 For example, Zhu et al. dispersed mechanisms of GQDs, their unique properties afford many
5 graphene oxide in dimethyl formamide (DMF) under sonication, applications in cellular and deep-tissue imaging. Sun and co-
and then transferred the suspension into Teflon autoclaves and 50 workers demonstrated the first bioapplication of nanographene

treated them at high temperature for a few hours to get GQDs oxide (NGO),162 i.e., single-layer graphene oxide sheets a few
with a QY of 11%.155, 156 Tetsuka and co-workers used the nanometers in lateral width. The PEGylated NGO sheets used
hydrothermal approach to treat graphene oxide sheets in ammonia were soluble in buffers and serum without agglomeration, and
10 solution to get GQDs with a QY between ~19 – 29%.157 The showed photoluminescence in the visible and infrared regions.
emission of GQDs can be tuned by controlling the hydrothermal 55 These NGO sheets had low background photoluminescence in the

temperature (Figure 15(a)), and the QYs can be further enhanced near-infrared (NIR) window. In addition, simple physisorption
to ~46% after modification with PEG. Wu et al. used a one-step through π-stacking was used to load the anticancer drug
pyrolysis of a natural amino acid (i.e. glutamic acid) to prepare doxorubicin onto NGO functionalized with antibody for selective
162
15 fluorescent GQDs with a QY of 54.5%.158 Recently, Dong and killing of cancer cells in vitro.
co-workers used L-cysteine as precursor to prepare S,N-co-doped 60 Compared with fluorescent carbon dots, GQDs can be used for
GQDs with a QY up to 73%,159 which is the highest value two-photon or multi-photon luminescence imaging.163, 164 Qian et
reported so far. al. used PEGylated graphene oxide nanoparticles to label HeLa
The preparation process significantly influences the optical cells,163 and observed that graphene oxide nanoparticles were
20 properties of GQDs. There are two types of emissions in GQDs, mainly localized in the mitochondria, endoplasmic reticulum,
i.e. intrinsic state emission and defect state emissions (Figure 65 Golgi apparatus, and lysosomes of HeLa cells with a two-photon

15(b)).160 The competition between these two states could be scanning microscope. They intravenously injected graphene
changed during preparation or post surface modification. For oxide nanoparticles into mouse bodies from the tail vein, and
example, Zhuo and colleagues oxidized graphene in concentrated observed their flow, distribution, and clearance in the blood
25 H2SO4 and HNO3, and then sonicated the mixture and calcinated vessels, utilizing a deep-penetrating two-photon imaging
it at 350 ˚C to remove acid.161 The resultant fluorescent GQDs 70 technique. These nanoparticles were also injected into the brains

did not exhibit excitation-dependent fluorescence [Figure 15(c- of gene transfected mice, and the in vivo two-photon
d)].161 However, Zhu et al. prepared green fluorescent GQDs luminescence imaging results showed that graphene oxide
through the hydrothermal approach.155 The green fluorescence nanoparticles were located at 300 µm depth in the brain,
 demonstrating the advantage of QGDs for deep imaging in
tissues. Recently, Liu et al. prepared nitrogen-doped GQDs as
efficient two-photon fluorescent probes.164 These N-GQDs
exhibited the highest two-photon absorption cross-section (up to
5 48000 Göppert-Mayer units) among the carbon-based materials.
They also demonstrated a large imaging depth of 1800 µm by a
study of penetration depth in tissue phantom (Figure 16).
In summary, surface-modified fluorescent carbon
nanomaterials (carbon dots and GQDs) have small size, Figure 17. (a) Schematic structure of multifunctional up-
10 distinctive photoluminescence properties, low toxicity, and low conversion nanoparticles; and (b) their potential applications
cost. These advantages offer them great potential for optical in bioimaging and therapy. Reproduced from Ref. 165.
imaging and biomedical applications, as they might gradually
replace conventional semiconductor QDs in these aspects. activator concentration, and accelerated sensitizer-activator
energy transfer rate arising from the decreased average minimum
60 distance between adjacent lanthanide ions. The high brightness
15 5 Ultra-small up-conversion nanocrystals
makes it possible to remotely track a single nanocrystal with a
Compared with previously mentioned fluorescent nanomaterials, microstructured optical-fibre dip sensor.178
up-conversion nanostructures, especially lanthanide-doped Ideal host materials should have low lattice phonon energy and
nanocrystals, have distinct advantages in fluorescence the minimum lattice mismatch with dopants (activators and
bioimaging, such as low autofluorescence background, large anti- 65 sensitizers). Rare-earth fluorides are generally chosen as host

20 Stokes shifts, sharp emission bandwidth, high resistance to materials, as rare-earth ions have similar ionic size and chemical
photobleaching, and high penetration depth and temporal properties to lanthanide ions, and their fluorides exhibit low
resolution,165-171 In addition, they can be used for multimodal phonon energy and high chemical stability. 166 In particular,
bioimaging and therapy (Figure 17). More bioapplications of up- NaGdF4 is extensively used as it can serve as a positive contrast
conversion nanoparticles can be found in recent reviews.165, 169-171 70 agent for magnetic resonance imaging (MRI). Johnson et al.

25 However, they usually have a larger size in comparison with prepared four different sizes of β-NaGdF4 nanoparticles between
those nanoprobes described previously (i.e. QDs, metal 2.5 nm and 8.0 nm.172 They found that the longitudinal relaxivity
nanoclusters, carbon dots, and GQDs). There are few reports on of nanoparticles increased from 3.0 mM-1s-1 to 7.2 mM-1s-1 with
ultra-small up-conversion nanoparticles, especially those below 5 decreasing particle size from 8.0 nm to 2.5 nm. The authors
3+
nm.172-177 Herein we mainly introduce the fundamentals of up- 75 doped Yb and Tm3+ into β-NaGdF4 to form 3.5 nm particles,
30 conversion nanoparticles and the progress in preparation and which exhibited an emission at 800 nm under the excitation of a
imaging application of ultra-small nanoparticles. 980-nm laser.172 Their results highlight the importance of
For up-conversion nanocrystals, their emission process preparation of ultra-small nanoparticles in order to achieve large
involves the sequential absorption of two or more photons, which relaxivity for MRI.
is fundamentally different from the multi-photon process, where 80 The fluorescence of up-conversion nanoparticles can be
35 the absorption of photons takes place simultaneously. There are engineered through modulation of activators, sensitizers, host
three types of up-conversion mechanisms, i.e. excited state materials, and their crystal phase, particle size, and surface
absorption (ESA), energy transfer up-conversion (ETU), and coating. Hasse and co-workers demonstrated the first example of
photon avalanche.166 The up-conversion nanocrystals usually multicolour emission of Yb3+/Er3+, and Yb3+/Tm3+ co-doped α-
179
consist of activators, sensitizers, and the host matrix [Figure 85 NaYF4 colloidal solution. In 2008, Wang et al. developed a
40 17(a)]. The activators should have more excited energy levels, general and versatile approach to fine-tune the multicolour
and the energy difference between each excited level and the emissions over a broad range with single wavelength
ground level should be close enough to facilitate photon excitation.180 By introducing Gd3+ during preparation, the authors
absorption and energy transfer in the up-conversion process. simultaneously controlled the crystal phase, particle size, and
Lanthanide ions such as Er3+, Tm3+, and Ho3+ have such ladder- 90 optical properties of the resultant nanocrystals.
181
Recently, a
45 like energy levels and are usually selected as activators. In order core-shell structure with a set of lanthanide ions incorporated into
to improve the luminescence efficiency, sensitizers are separated layers was designed. The core-shell structure can
introduced. Yb3+ is usually chosen as sensitizer because it has minimize the deleterious effects of cross-relaxation. The bright
only one excited energy level (2F5/2), and the transition between up-conversion emission was achieved through Gd 3+ mediated
the ground level (2F7/2) and excited level is strongly resonant with 95 energy migration without long-lived intermediate energy
50 many f-f transitions of lanthanide ions. The concentration of states.182, 183
activators, and the molar ratio between activators and sensitizers In up-conversion nanoparticles, minimizing the depletion of
is usually kept low to avoid the quenching effect.166 Zhao et al., excitation energy is the key to tuning their luminescence. The
however, showed that up-conversion luminescence can be excitation energy can randomly migrate from an atom to its
significantly enhanced by using much higher activator 100 neighbouring atoms that are isotropically distributed in a 3D
3+
55 concentrations (e.g. 8 mol% Tm in NaYF4) under relatively structured crystal sublattice (type I in Figure 18). This energy can
178 also migrate in a crystal with a 2D layer structure (type II), or in a
high-irradiance excitation. The authors attributed the high
brightness to a combination of high excitation intensity, increased crystal featuring a 1D atomic chain structure (type III).184
 Figure 20. Biodistribution of 5.1nm (NaGdF4) and 18.5nm
(NaGdF4:Yb,Er) nanoparticles in different organs and tissues
of mice. Reproduced from Ref. 189.
NaYF4:Yb3+/Tm3+@NaGdF4:Yb3+ nanoparticles were first
prepared by complex thermal decomposition method, followed by
Figure 18. Schematic illustration of the topological energy surface modification and conjugation with the trans-platinum
migration pathways in different types of crystal sublattice. 30 (IV) pro-drug. The up-conversion nanoparticles can not only
Reproduced from Ref. 184. deliver the platinum (IV) pro-drugs into the cells effectively, but
convert near-infrared light into UV to activate pro-drug as well.
Meanwhile, they can further serve as contrast agents for
multimodality imaging to guide cancer treatment. The pro-drug-
35 conjugated nanoparticles under near-infrared irradiation led to
better inhibition of tumor growth than that under direct UV
irradiation in the mouse test.186 Such multifunctional up-
conversion nanoparticles have been a subject of intensive
research due to their potential in disease diagnosis and
Figure 19. Multifunctional upconversion nanoparticles for 40 treatment.165, 170
diagnosis and treatment of cancer through imaging-guided For in-vivo bioapplications, one of the major issues for up-
therapy. Reproduced from Ref. 186. conversion nanocrystals is the fate of nanoparticles and potential
toxicity of lanthanide ions.187, 188 Liu et al. prepared 5.1 nm
Recently, Wang et al. proposed that migration of the excitation NaGdF4 and 18.5 nm NaGdF4:Yb/Er nanoparticles with the same
energy can be effectively minimized through use of a type IV 45 surface modification and investigated their biodistributions in
(Figure 18) lattice containing arrays of isolated atomic clusters.184 different organs and tissues of mice (Figure 20). 189 The
This allows to minimize the concentration quenching of the accumulation of both types of nanoparticles in liver decreased
5 luminescence, and generates an unusual four-photon-promoted with the circulation time. In contrast, their accumulation in spleen
violet up-conversion emission from KYb2F7:Er (2 mol%) with an increased with the circulation time. This suggests that both of
intensity more than eight times higher than that previously 50 these nanoparticles may be eliminated through the biliary
reported.184 The approach of enhancing up-conversion through pathway. Analysis of urine collected at different time points
energy clustering at the sublattice level may provide new indicates that renal clearance was one of the major elimination
10 opportunities to engineer up-conversion nanoparticles for diverse pathways for 5.1-nm particles, but not for 18.5-nm particles.
applications. Further analysis on faces by TEM shows that these particles do
The good understanding of the energy migration, luminescence 55 not change in shape and size, suggesting the high stability of up-
mechanism, and the recent advances in wet chemistry have conversion nanoparticles in vivo.189
enabled the fine-tuning of particle size (even in the small size Although up-conversion nanoparticles do not show acute
15 range), crystal structure, surface functionalities, and optical toxicity at the cell or animal level, it is necessary to investigate
properties of up-conversion nanocrystals for bioimaging, drug their long-term toxicity. Another drawback of up-conversion
delivery, and sensing.165, 167, 169 Their fluorescence has been 60 nanocrystals is the low quantum yield (usually less than 1%) in
applied to image cells and small animals.185 As mentioned comparison with other fluorescent agents. 165 Preparation of
previously, Gd-based up-conversion nanoparticles are particularly highly efficient up-conversion nanocrystals remains a great
20 interesting as they can serve as fluorescent nanoprobes and challenge.
contrast agents of MRI simultaneously. Recently, a
multifunctional drug delivery system combining up-conversion
luminescence/magnetic resonance/computer tomography 65 6 Fluorescent silicon nanoparticles
trimodality imaging and NIR-activated platinum pro-drug Fluorescent silicon nanoparticles (Si NPs) have also attracted
25 delivery has been developed by Dai and co-workers (Figure considerable attention in bioapplications due to their excellent
19).186 Organic-soluble core−shell biocompatibility as silicon naturally exists in human body as a
 Figure 21. (a) Silicon nanoparticle fractions under ambient
light and under photoexcitation at 365 nm; (b-c) size-
dependent absolute QYs of Si nanoparticles. Reproduced
from Ref. 191.
trace element.18, 20, 190 More importantly, they have tunable
fluorescence from visible to near-infrared window. Compared
with other semiconducting QDs, the preparation of high-quality
water-soluble and biocompatible Si QDs is devious and
5 laborious. Colloidal Si NPs are conventionally prepared by

etching annealed SiOx with HF, plasma approach,

electrochemical method, laser ablation, reduction of SiCl4, and
solvothermal reaction of sodium silicide with NH4Br.18, 190-193
These Si NPs are usually functionalized with hydrophobic ligands
10 such as styrene, alkyl, and octene. They are photochemically

stable in non-polar solvents up to 1 year.192 For example, high-

temperature thermal processing of the sol-gel precursor derived
from trichlorosilane (HSiCl3) produced Si NPs embedded within
the SiO2 matrix.191, 194 Si NPs were released after etching the SiO2
15 matrix with HF, and then passivated with allylbenzene through

the thermally initiated hydrosilylation reaction. The resultant

colloidal Si NPs were fractionated by size selective precipitation Figure 22. (a) Photostability of Si NPs in comparison with
to obtain monodisperse nanoparticles, which showed strong FITC, CdTe QDs and CdSe/ZnS QDs; (b) cell nuclei are
quantum confinement effects and size-dependent absolute QYs labeled by Si NPs (Left), microtubules are labeled by FITC
20 (Figure 21).
191
The absolute QYs increased with particle size up (middle); and superposition of the two fluorescence images
to 43%. (right); (c) time-dependent stability comparison of
fluorescence signals of Hela cells labeled by Si NPs (blue)
During preparation, Si NPs can be chemically doped to
and FITC (green). Reproduced from Ref. 204.
introduce other functions.195, 196 Paramagnetic fluorescent Si NPs
were prepared by solvothermal decomposition of Mn-doped somewhat independent of particle size.197, 198 Recently, Dasog et
195
25 sodium silicide. The resultant Mn-doped Si NPs showed a 40 al. prepared Si NPs using three most widely cited procedures (i.e.

longitudinal relaxivity (r1) of 25.50 ± 1.44 mM-1s-1 and a etching of annealed SiOx, reduction of SiCl4, and solvothermal
transverse relaxivity (r2) of 89.01 ± 3.26 mM-1s-1 under a reaction of sodium silicide with NH4Br), 197 and found their
magnetic field of 1.4 T at 37 °C. Similarly, Fe-doped Si NPs were conversion of red-fluorescence to blue emission. Their findings
prepared and exploited as bimodal imaging agents.196 The use of suggest that the presence of trace nitrogen and oxygen even at the
30 reactive sodium silicide makes their control preparation difficult 45 ppm level in Si NPs gives rise to the blue emission, and support

and could limit their broad applications, and thus development of the hypothesis that the nitrogen defect or impurity site contributes
novel preparation approaches is necessary. to the blue emission.197
Similar to carbon dots and GQDs, Si NPs produced from In order to apply Si NPs to bioimaging, tremendous efforts
different methods seem identical, but their optical properties are have been made to prepare water-soluble and biocompatible Si
199-205
35 dramatically different. For example, the Si NPs prepared with 50 NPs through simple and efficient methods. For example, Si
high-temperature method routinely exhibit photoluminescence NPs with excellent aqueous dispersibility, robust photo- and pH-
agreeing with the effective mass approximation (EMA), while stability, strong fluorescence (∼15%), and favorable size (∼4 nm)
those prepared via solution methods exhibit blue emission are facilely and rapidly prepared from Si nanowires and glutaric
 acid in a short reaction time (e.g., 15 min) by He and co-
workers.202 These Si NPs are particularly suitable for long-term
and real-time cellular imaging due to their higher photostability
than II-VI QDs and dyes (e.g. CdTe QDs and FITC). Distinctive
5 red fluorescence of Si NPs can be retained throughout 240-min

irradiation. In contrast, the green fluorescence of FITC rapidly

diminishes in 3 min due to severe photo bleaching, and the red
signals of CdTe QDs nearly vanishes after 25-min irradiation.
The MTT assays showed negligible cellular toxicity to HeLa
10 cells, demonstrating the excellent biocompatibility of Si NPs.

Furthering their research, the authors used (3-

aminopropyl)trimethoxylsiliane as precursor and prepared 2.2 nm
Si NPs by the similar method.204 The obtained Si NPs exhibited
strong green fluorescence with a QY of 20-25%,
15 biocompatibility, and robust photo- and pH-stability. As shown in

Figure 22, FITC, CdTe and CdSe/ZnS QDs, and Si NPs exhibited
distinct fluorescence behaviors during initial UV irradiation.204
The fluorescent signals of the former three samples were
gradually reduced with increasing irradiation time. The
20 fluorescence of FITC was completely quenched within 15 min

irradiation. In contrast, the Si NPs preserved stable and bright

fluorescence during long-time (e.g., 180 min) irradiation under
the same conditions. Figure 22c also shows that Si NPs-labelled
nuclei (blue) and the FITC-labelled cellular microtubules (green)
25 were intense and clearly spectrally resolved, respectively. The Si

NP-labelled nuclei retained stable fluorescence during

observation for 60 min, however the fluorescence from FITC
labels drastically decreased in 3 min due to severe photo
bleaching.
30 In addition to in vitro labelling cells, Si NPs can also be used
in multiple cancer-related in vivo applications, including tumor
vasculature targeting, sentinel lymph node mapping, and
multicolor NIR imaging in live mice.206 Erogbogbo and
coworkers demonstrated that Si NPs can overcome dispersibility Figure 23. (Top) biodistributions of Si NPs in mice assessed
35 and functionalization challenges for in vivo imaging through by ICP-MS analysis, fluorescence images of frozen tissue
surface functionalization, PEGylated micelle encapsulation, and sections, confocal microscopy images; (bottom) histological
bioconjugation process, which produced bright, targeted images of (a) brain, (b) heart, (c) liver, (d) spleen, (e) lung, (f)
nanospheres with stable luminescence and long (>40 h) tumor kidney, (g) lymph, (h) intestine, and (i) skin harvested from
accumulation time in vivo. Recently, the biodistribution and rhesus macaques administrated with Si NPs. Reproduced
40 toxicity of Si NPs in mice and monkeys have been assessed from Ref. 207.
(Figure 23).207 The top images in Figure 23 show the evaluated by in vivo positron emission tomography (PET)
biodistribution of Si NPs in mice, the fluorescence image of 60 imaging and ex vivo gamma counting.208 A new macrocyclic
frozen tissue sections, and the confocal images, which clearly ligand-64Cu2+ complex was conjugated with dextran-coated Si
reveals particles localized in the liver, spleen, and kidneys after NPs and served as PET agent. The results show that conjugates
45 injection. The ICP-MS data show notable increase of silicon were excreted via renal filtration shortly post injection and also
levels in the liver, spleen, lung, kidneys, and lymph. The accumulated in the liver, again demonstrating the stability and
concentration of silicon in the lymph and kidneys declined over 65 biocompatibility of Si NPs.
the 14-week time period, while the liver and spleen retained a In summary, Si NPs have tuneable fluorescence from visible to
significant fraction of the silicon injected, even after 14 weeks.207 near-infrared window, excellent biocompatibility, chemical, and
50 There is no evidence of the biodegradability of silicon NPs. The photostability. These properties make them very attractive in
bottom images in Figure 23 display the histological images of the bioimaging, however long-term studies on their safety and
brain, cerebellum, atrium, ventricle, heart muscle, lung, kidney, 70 adverse effects are still needed for their clinical applications.
liver, spleen, renal tubule, intestine, lymph nodes, and skin of the
rhesus macaques.207 There was no sign of nanoparticle-induced 7 Summary and outlook
55 changes in these organs and tissues. This research indicates We have summarized the current state of the art on ultra-small
neither mice nor monkeys showed overt signs of toxicity inorganic nanoparticles for fluorescence bioimaging. These ultra-
reflected in their behavior, body mass, or blood chemistry. The small nanoparticles bridge the gap between big particles and
biodistribution of Si NPs in mice was also quantitatively 75 molecules. They have unique properties and great potential in
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