Journal of Medical and Biological Engineering, 29(6): 276-283 276

Review:

Synthesis of Fluorescent Metallic Nanoclusters toward

Biomedical Application: Recent Progress and

Present Challenges

Cheng-An J. Lin1,2 Chih-Hsien Lee1,2 Jyun-Tai Hsieh1,2 Hsueh-Hsiao Wang3,4

Jimmy K. Li5 Ji-Lin Shen6 Wen-Hsiung Chan7 Hung-I Yeh3,4 Walter H. Chang1,2,*

1Department of Biomedical Engineering, Chung Yuan Christian University, Chungli 320, Taiwan, ROC 2Center for Nano Bioengineering, Chung Yuan Christian University, Chungli 320, Taiwan, ROC

3Departments of Medical Research and Internal Medicine, Mackay Medical College, Mackay Memorial Hospital, Taipei 252, Taiwan, ROC 4Department of Medicine, Mackay Medical College, Mackay Memorial Hospital, Taipei 252, Taiwan, ROC

5Department of Material Science, National University of Tainan, Tainan 700, Taiwan, ROC 6Department of Physics, Chung Yuan Christian University, Chungli 320, Taiwan, ROC

7Department of Bioscience Technology, Chung Yuan Christian University, Chungli 320, Taiwan, ROC

Received 5 Oct 2009; Accepted 20 Nov 2009

Abstract

Recent advances in nanomaterials have produced a new class of fluorescent labels by biocognition molecules to

fluorescent noble-metal nanoclusters such as Au and Ag. In particular, the emission wavelength of metallic

nanoclusters can be tuned by changing the capping molecules, and a single light source is needed for simultaneous

excitation of all different-emissive nanoclusters, which is similar to semiconductor quantum dots. In this review, we

highlight the recent advances in synthesis approaches, biomolecular conjugation and its biomedical application.

Fabricating the color-emitting metal nanoclusters using the template-based synthesis (i.e., dendrimer, oligonucleotide,

proteins, polyelectrolyte, and polymer) and monolayer-protected nanocluster (MPC) synthesis (i.e., dihydrogen lipoic

acid and mercaptoundecanoic acid) are described. High-quality nanoclusters are also more biocompatible and stable

against photobleaching compared with organic dyes. These novel optical properties render the fluorescent noble-metal

nanoclusters ideal fluorophores for multicolor and multiplexing applications in biomedical engineering and molecular

biotechnology.

Keywords: Fluorescent gold nanoclusters, Fluorescent silver nanocluster, Quantum dots, Synthesis, Bioconjugation,

Cell labeling, Detection, Imaging

1. Introduction

Noble metal nanoclusters (e.g., Au, Ag) typically possess

sizes below 2 nm and have been attracting attention for their

unique role in bridging the “missing link” between atomic and

nanoparticle behavior [1]. Recent studies have focused on their

quantum electronic properties including chirality [2-5],

ferromagnetism [6], photoluminescence [7-14], quantum

behavior [15-18], single-molecule optoelectronics [19,20],

sensing [21] and bioassay [22,23]. Analogous to semiconductor

* Corresponding author: Walter H. Chang

Tel: +886-3-2654503; Fax: +886-3-2654581

E-mail: whchang@cycu.edu.tw

quantum dots containing strong quantum-size confinement

when particle sizes are smaller than the exciton Bohr radius

(about 4-5 nm for CdSe) [24], gold nanoparticles show a

size-dependent plasmon absorption band when their

conduction electrons in both the ground and excited states are

confined to dimensions smaller than the electron mean free

path (ca. 20 nm) [25], but plasmon absorption disappears

completely for nanoparticles less than 2 nm which Mie’s

theory no longer can be applied [26-28]. Interestingly, metal

nanoclusters confined to a second critical regime having sizes

comparable to the Fermi wavelength of the electron (ca. 0.7

nm), which results in molecule-like properties of discrete

electronic states [15,29-31] and size-dependent fluorescence

[1,21] (i.e., a scale function of the number of atoms within the

J. Med. Biol. Eng., Vol. 29. No. 6 2009 277

cluster from the energy differences between the highest

occupied molecular orbital (HOMO) and the lowest

unoccupied molecular orbital (LUMO). The photoluminescent

properties are attributed to the recombination involving d-band

excitation, as described in Figure 1 [29].

The semiconductor quantum dots (QDs) have already

become a new class of fluorescent labels [32,33] due to their

unique optical properties as well as offering potential

invaluable benefits such as cancer targeting [34] and

biomedical imaging [35,36]. However, the heavy metals

contained in QDs are toxic, making them unsuitable for in vivo

clinical application, and may pose risks to human health as

well as the environment under certain conditions [37]. In

contrast to QDs, noble metal nanoclusters (NCs) are highly

attractive for bioimaging and biolabeling applications due to

their low toxicity as well as its ultra fine size. Recently, the

increasing use of metal-containing compounds in therapy and

diagnosis [38] have made possible the advance of metal

nanoclusters as an alternative building block of biomedical

probes using their luminescent properties.

Emission

Absorption

Wave number

Emission

Absorption

Figure 1. A simple energy diagram of photoluminescence in gold

nanoclusters involving d-band excitation. Absorption of a

photon promotes an electron from the narrow d band to the

empty sp band above the Fermi level. After some carrier

relaxation, radiative recombination responsible for the

emission then occurs between an electron (probably one

near or below the Fermi level) and the excited hole,

resulting in the visible-near-IR emission.

2. Experimental observations

The early findings on photoluminescence (PL) of noble

metals were in bulk metals [39], roughened surface [40]

experiments and a detailed theoretical study [29]. However,

luminescent properties of metal clusters [41,42], especially the

water-soluble ones [43], attracted great attention in the 1990s.

Metal nanoclusters have also gained great improvement in

synthesis strategies which can be divided into two categories:

the template-based synthesis and monolayer-protected

nanoclusters (MPCs) synthesis. Researchers reported the

successful synthesis of fluorescent Au or Ag nanoclusters with

high quantum yield using poly(amidoamine) dendrimers [1,13]

and DNA [44,45] as size-confining template. Nanoclusters

sizes fit with emission energies via the simple relation,

EFermi/N1/3, predicted by the spherical jellium model [1].

Emission energy of the number of atoms, N, in each gold

nanocluster can be predicted by the simple scaling relation, in

which EFermi is the Fermi energy of bulk gold. Emission energy

decreases with increasing number of atoms. Other examples,

such as polymer microgel [46], multi-arm star copolymer [47],

polyelectrolyte [48,49] and organic-inorganic hybrids [50],

also show that template-based synthesis has proven to be a

simple method. However, some existing factors raise concerns

when involving further bioprobe designs, e.g., the relatively

small Stoke’s shifts [1,20], hard-defined core-shell structures

[46,49,50], large matrix size [22,46] and the containment

non-fluorescent nanoparticles during synthesis [1,13,48].

Large fluorescent tags, for example, can perturb the labeled

biomolecules and cause artificial movement within the cell.

Since Brust et al. [51] developed the first preparation of

hydrophobic MPCs, the PL quantum yield of thiolated MPCs,

such as gold nanoclusters protected by glutathione [7,11,14,52],

tiopronin [8,53,54], meso-2,3-dimercaptosuccunic acid [55],

phenylethylthiolate [30], dodecanethiol [12], and

mercaptoundecanol [21,23] have been enhanced by several

orders of magnitude to visible level with respect to that of bulk

gold (10-10). It contains large Stoke’s shifts, and even tunable

fluorescence via selection of appropriate length of thiolated

ligands [21]. Furthermore, the molecular structure of MPCs can

also be resolved by several advanced techniques such as mass

spectrometry [52] and X-ray crystallography [56,57] on the basis

of purification techniques, e.g., crystallization [58],

chromatography [59], and electrophoresis [2,14,52]. Although

the thiolated MPCs offer great potential, examples [22,23] of

their application to biomedical research using its fluorescent

properties are rare. Even though gold nanoparticles are

biocompatible, the knowledge and approaches from synthesis to

bio-probe design have been limited.

3. Typical examples of synthesizing highly fluorescent

metallic nanoclusters

Color-emitting fluorescent metallic nanoclusters has been

developed recently [1,21,45,60]. However, there is no general

route to fabricating high-quality nanoclusters emitters. The

emission wavelength not only correlates with its size but also

its protected molecules (Figure 2). This review introduces

several reproducible synthesis methods toward promising

biomedical applications.

3.1 Blue-emitting gold nanoclusters

Researchers reported the creation of quantum-confined,

water-soluble, high quantum yield Au nanoclusters (Au8)

embedded in poly(amidoamine) dendrimer (PAMAM) which

is repeatedly branched molecules with different generations

[1,13]. The general procedure consists of mixing dendrimer

(G4-OH or G2-OH) and gold ions (HAuCl4.nH2O) in distilled

water. After adding strong reduction agent of NaBH4, small

fluorescent Au nanoclusters (i.e., dendrimer-encapsulated

nanoclusters) and large nanopart icles are created

simultaneously. The confined intra-space of dendrimer restricts

the growth of gold nanoclusters, but further purification

through centrifugation is usually required to remove the large

Fluorescent Metallic Nanoclusters toward Biomedicine 278

Figure 2. Representative fluorescent noble-metal nanoclusters scaled as a function of their emission wavelength superimposed over the spectrum.

Protected molecules show different capabilities to tune the emission wavelength of metallic nanoclusters from current reports.

gold nanoparticles. Recently, Martinez et al. reported

nanoparticle-free synthesis of fluorescent gold nanoclusters via

a mild biologically derived reductant (i.e., vitamin C) [61].

The blue-emitting gold nanoclusters can also be produced

without additional reductant. The mixture of gold

precursor/dendrimer stock solution is only incubated at 37°C

for 3 days to form blue-emitting gold nanoclusters

(Au8@PAMAM) which infer to being reduced by the presence

of the surface hydroxyl group. In recent examples, the

blue-fluorescent gold nanoclusters with high quantum yield

(QY > 35%) have been mostly produced by using a dendrimer

as template.

3.2 Green-emitting gold nanoclusters

Although appropriate mixture of PAMAM dendrimer and

HAuCl4 in distilled water can form green-emitting gold

nanoclusters upon adding reductants (i.e., NaBH4, ascorbic

acid), some disadvantages such as low yields [1] or containing

mixtures [61] present in using those dendrimers as template.

Huang et al. started to modify small gold nanoparticles with

various alkanethiol ligands to control the luminescence

properties [21]. In brief, green-emitting gold nanoclusters

(λem max = 500 nm, QY ≈ 4%) could directly form by adding

the mercaptoundecanoic acid (MUA) into the prepared small

gold nanoparticle solution (THPC-AuNP, ~3.4 nm), which

were synthesized through reduction of HAuCl4 with THPC

(tetrakis(hydroxymethyl) phosphonium chloride). After the

thiol group of MUA replaced the THPC on the gold

nanoparticle, the nanoparticles became small and fluorescent

nanoclusters (AuNC@MUA). Changing the THPC-to-HAuCl4

concentration ratios could tune the emissive wavelength from

green (1.0) to yellow (2.0) without losing quantum yield, as

shown in Figure 3. This key advance might be attributed to the

greater decrease in size from the THPC-AuNP than that from

NaBH4-reduced AuNP [62].

3.3 Red-emitting gold nanoclusters

Most thiol-related MPCs, such as gold nanoclusters

protected by glutathione [7,11,14,52], tiopronin [8,53,54],

meso-2,3-dimercaptosuccunic acid [55] and phenylethylthiolate

White light UV excitation

Figure 3. Four distinguishable emission colors of fluorescent gold

nanoclusters excited with a UV lamp. Upper left illustrates

the possible structures of AuNC@PEI, AuNC@MUA and

AuNC@DHLA. Upper right is SEM image of

AuNC@DHLA. Lower figures: From left to right (blue to

red) which is made in PEI (a), MUA (b,c) or DHLA (d)

respectively, the emission maximum are located at 450, 500,

550, and 650 nm.

[30], emit wavelength range from red to infrared, but have low

quantum yields (QY < 1%). Recent advances exhibit decent

improvement on red-emitting gold nanoclusters by using either

an organic phase [63] or an aqueous phase [64] route. Lin et al.

created a precursor-induced nanoparticle etching technique to

fabricate one-pot nanoclusters, which become highly

red-luminescent (λem max = 650 nm, QE ≈ 4%) upon ligand

exchange [63]. In general, gold nanoparticles prepared from a

single-phase reaction [65] either with or without purification

by methanol precipitation are etched into small nanoclusters by

the gold precursor (AuCl3 or HAuCl4) solution. The etched

gold nanoclusters lose their surface plasmon resonance

properties and lead to a yellowish or even colorless transparent

solution, whereas the original larger gold nanoparticles possess

strong surface plasmon absorption around 520–530 nm. The

addition of freshly reduced lipoic acid (DHLA) can replace the

surfactants on the etched gold nanoclusters via the formation

of strong dithiol-Au bonds, whereby the acid headgroup points

towards the solution. Upon such ligand exchange with lipoic

acid, the gold nanoclusters (AuNC@DHLA) become

J. Med. Biol. Eng., Vol. 29. No. 6 2009 279

red-emitting fluorophores. By deprotonization under basic

buffer, the etched gold nanoclusters become water-soluble and

form a mono-dispersion stabilized by electrostatic repulsion

(Figure 3).

Recently, Xie et al., the second example, created another

novel “green” synthetic route for the preparation of

red-emitting gold nanoclusters (λem max = 640 nm, QE ≈ 6%)

produced using bovine serum albumin (BSA) as template at

the physiological temperature [64]. They supposed the process

was similar to a biomineralization behavior of organisms in

nature, i.e., the functional proteins provide scaffolds for

mineral forms upon sequestering and interacting with

inorganic ions. Briefly, the BSA molecules sequestered gold

ions and entrapped them upon addition of Au(III) ions to the

aqueous protein solution. The entrapped ions undergo

progressive reduction to form gold nanoclusters in situ when

activating the reduction ability of BSA molecules by adjusting

the reaction pH to ~12. Each gold nanocluster of 25 gold

atoms was stabilized within BSA molecules, which possess

good biocompatibility as well as postsynthesis surface

modification with functional ligands.

3.4 Oligonucleotide-stabilized silver nanocluster fluorophores

The high affinity of Ag+ for cytosine based on ssDNA has

enabled creation of short oligonucleotide-encapsulated Ag

nanoclusters without formation of large nanoparticles. Using

oligocytosine scaffolds, silver nanocluster emitters have been

created but in highly heterogeneous mixtures containing at least

four inseparable species [66,67]. Recently, Richards et al.

reported on five distinct silver nanoclusters of spectral pure

emitters encapsulated in single-strand DNA, offering a

convenient scaffold to tune the emission throughout the visible

and near-IR spectrum [45]. In general, a 6:1 molar ratio of silver

ions to oligonucleotide was mixed, followed by NaBH4 reduction

to form silver nanoclusters. The emission of fluorescent silver

nanoclusters could be programmed by DNA sequences, i.e., blue

emitters created in 5’-CCCTTTAACCCC-3’, green emitters

created in 5’-CCCTCTTAACCC-3’, yellow emitters created in

5’-CCCTTAATCCCC-3’, red emitters created in

5’-CCTCCTTCCTCC-3’, and near-IR emitters created in

5’-CCCTAACTCCCC-3’. The investigators also found the

lengthened oligocytosine (5’-AATTCCCCCCCCCCCCAATT-3’

(C12) at pH 7) further improved the quantum yield by 30% for

the yellow emitter. Excepting the blue and green emitting

species, which lack of photostability, the yellow, red, and

near-IR emitting species show great potential in single-molecule

bio-labeling.

3.5 Polyelectrolyte-stabilized silver nanocluster fluorophores

Photoreduction has been proved to be an effective method

for preparing Ag nanoclusters. Kumacheva et al. firstly

reported the successful photogeneration of fluorescent Ag

nanoclusters using polymer microgel [46]. Shen et al. then

prepared fluorescent Ag nanoclusters using multi-arm star

copolymers as templates [47]. However, several synthetic

issues remain in these methods, such as complicated

preparation of template or the simultaneous formation of larger,

non-fluorescent nanoparticles. Shang and Dong [49] recently

found that a common polyelectrolyte poly (methacrylic acid)

(PMAA) offers several crucial advantages as a template:

(a) negative-charged carboxylic acid can coordinate with Ag+

ions, (b) hydrophobic region of methyl group facilitate the

formation of Ag nanoclusters, and (c) application is easily

extended into areas such as biomedicine. A freshly prepared

mixture solution of AgNO3 (0.05 M) and PMAA (0.1 M) was

incubated in the dark for 10 mins, then subjected to

UV-irradiation at 365 nm for appropriate time intervals. The

resulting solution was observed in obvious color changes from

colorless to dark red. The quantum yield of as-prepared Ag

nanoclusters was 18.6%. The emission maximum of Ag

nanoclusters was found to shift significantly to longer

wavelength (610 to 660 nm) with increasing excitation

wavelength (450 to 580 nm).

4. Bioconjugation

Bioconjugation can provide extra functionality onto

nanoclusters [68], such as stability, biocompatibility, and

targeting, by using reactive functional groups of primary amines,

carboxylic acids, alcohols, or thiols. Most MPC-based

fluorescent gold nanoclusters are protected by the carboxylic

acids, which can conjugate with amino-molecules to form stable

amide bond catalyzed with a carbodiimide or sulfo

N-hydroxysuccinimide ester. A simple approach has recently

been used by Lin et al. [63] in which polyethylene glycols amine

(PEG-NH2) streptavidins and albumins can be covalently

conjugated onto carboxylic fluorescent gold nanoclusters by

1-[(3-dimethylamino)-propyl]-3-ethylcarbodiimide hydrochloride

(EDC) activation. Using high binding affinity between thiol and

gold, Huang et al. [69] could directly prepare fluorescent

mannose-protected Au nanoclusters via addition of

11-mercapto-3,6,9-trioxaundecyl-α-D-mannopyranoside (Man-SH)

onto the surface of as-prepared gold nanoparticles. Similarly,

dendrimer-encapsulated gold nanoclusters can be modified

carboxylic acid by ligand exchange with MUA [70], followed by

conjugating the nuclear localization signal (NLS) peptide upon

EDC activation. Besides the covalent conjugation, proteins can

also be directly adsorbed onto negatively charged nanoclusters

through electrostatic interaction. For example, platelet-derived

growth factor, which is a breast cancer marker protein, has been

readily conjugated to green-emissive gold nanoclusters through

electrostatic and hydrophobic interaction [23].

In addition, fluorescent metallic nanoclusters could also

directly synthesize onto the biological template without further

bioconjugation steps. The ssDNA scaffold affords a single

point of attachment, while simultaneously stabilizing the

few-atom, strongly emissive Ag nanoclusters [20,71].

Nucleolin, one of the major proteins to bind silver atoms in

silver staining [72], appears to nucleate the formation of

fluorescent Ag nanoclusters [73]. BSA is used to sequester and

reduce the Au precursors in situ to form red-emitting Au

nanoclusters. In these cases, either oligonucleotides or proteins

have already provided a biocompatible interface upon

nanocluster formation. The above bioconjugation techniques

are illustrated in Figure 4.

Fluorescent Metallic Nanoclusters toward Biomedicine 280

Figure 4. Schematic illustration of bioconjugation methods. (a) Use of

a bifunctional ligand such as mercaptoundecanoic acid

derivative for linking fluorescent Au nanoclusters to

biomolecules; (b) positively charged biomolecules are linked

to negatively charged nanoclusters by electrostatic attraction;

(c) covalent linkage of amide bond via EDC chemistry; (d)

oligocytosine encapsulation of fluorescent Ag nanoclusters.

5. Cellular labeling

Because the fluorescent metallic nanoclusters have decent

quantum yield and photostability, they can be used as cell

markers for long-term studies such as cell-cell interactions,

cell differentiation, and tracking. The idea to use fluorescent

metallic nanoclusters is based on the discovery that

nanoclusters can be internalized by cells, by either

receptor-mediated [70] or nonspecific endocytosis [63].

Blue-emitting Au nanoclusters functionalized with site-specific

leading peptide such as SV40 NLS can enter the cytoplasm of

living HeLa cells, where the non-functionalized ones shows no

intracellular signalings for 1.5 hr treatment [70]. Human aortic

endothelial cells can uptake the un-modified red-emitting Au

nanoclusters (AuNC@DHLA) after around 5 hr incubation [63]

as shown in Figure 5. But the exact pathway of incorporating

metallic nanoclusters into cells under endocytosis is not

understood, requiring further investigation.

Figure 5. Delivery of AuNC@DHLA in human aortic endothelial cells.

The uptake of gold nanoclusters, shown in red fluorescence

were examined using fluorescence microscopy. The blue

fluorescence is nucleus counterstained with bisbenzimide.

After 4 hours of AuNC@DHLA delivery, medium was

replaced with fresh culture medium and fluorescence images

(b) were acquired after 44 hours of recovery. Control (a),

cells without any treatment. Bar, 50 µm.

Labeling the fixed cells using organic dyes or

semiconductor quantum dots is prevalent in biomedical

research [74]. Recently, fluorescent Au nanoclusters have been

used to label the endogenous biotin which is widely distributed

in the body, especially in kidney, liver and brain. The

endogenous biotin of paraformaldehyde-fixed human

hepatoma cells (GepG2) can be marked by

streptavidin-conjugated AuNC@DHLA, fitted with the

positive-stained group with FITC-streptavidin. Other cells

could also be labeled with avidin-conjugated Ag nanoclusters

upon general membrane biotinylation [71]. As non-specific

labeling also occurs in fluorescent metallic nanoclusters, it can

be eliminated by PEGylation [63]. Contrary to direct labeling

using functionalized nanoclusters, Dickson et al. [75] created

another novel labeling method for the fixed cells, i.e., the

shuttle-based fluorogenic silver-cluster biolabels. Silver ions

are first complexed with 3-(2-aminoethylamino)

propyltrimethoxy silane before poly(acrylic acid) (PC) and

borohydride reduction to form low quantum yield nanoclusters

(dark, QY ≈ 3%). PC-modified nanoclusters readily transfer

nanoclusters to high-affinity ssDNA sequence, resulting in

high quantum yield nanoclusters (bright, QY ≈ 30%). After

cellular actins are stained with anti-actin/C12 conjugates, the

actins could be then fluorescent marked by incubating with

PC-modified Ag nanoclusters. Only nanoclusters/C12

conjugates appears brightly fluorescent signals.

6. Fluorescent nanoclusters as biosensors

Recognition-based biosensors capable of specifically

detecting chemical and bio-agents in their environment are under

active development using semiconductor dots, but seldom

employing fluorescent noble-metal nanoclusters, owing to

limited knowledge of their optical physics. Fluorescent gold

nanoclusters (AuNC@MUA) were firstly used to sense mercury

(II) based on fluorescence quenching through Hg(II)-induced

aggregations of AuNC@MUA [21]. Similarly,

glutathione-protected gold nanoclusters (AuNC@GSH) were

highly sensitive to the essential biological metal ions Cu2+ based

on aggregation-induced fluorescent quenching [76]. However,

an important goal for biosensors is the capability of continuously

monitoring concentrations of specific targets in a simple and

reliable manner. Huang et al. [23] developed new competitive

homogeneous photoluminescence quenching assay for analyzing

proteins using bioconjugated photoluminescent Au nanodots as

donors and bioconjugated spherical Au nanoparticles as

accepters. Aptamer (Apt), an oligonucleotide, has a higher

affinity for specific proteins such as a breast cancer marker

protein, platelet-derived growth factor (PDGF). The

PDGF-modified fluorescent Au nanoclusters can attach onto the

Apt-modified gold nanoparticles, consequently resulting in

fluorescence quenching (“off” state) through resonance energy

transfer. Addition of free PDGF can further recover the

fluorescence of PDGF-modified nanoclusters through the

competitive binding, releasing the quenched nanocluster to “on”

state. Another example of sensing proteins, fluorescent

mannose-protected Au nanoclusters (AuNC@Man) are capable

of sensing concanavalin A with high sensitivity and remarkable

selectivity over other proteins and lectins [69]. AuNC@Man

also has the capability of binding mannose-specific adhesion

FimH of type 1 in E. coli bacteria, yielding brightly fluorescent

cell clusters.

J. Med. Biol. Eng., Vol. 29. No. 6 2009 281

7. Conclusion and perspectives

This has been a brief overview of many efforts that have

been employed in the development of fluorescent noble-metal

nanoclusters, which present promising application in biomedical

fields. Similar to semiconductor quantum dots [77], there have

been a good example for the general paradigm underlying

nanobiotechnology in the past decade. Synthetic breakthroughs

of fluorescent metallic nanoclusters have opened the exploitation

of biomedical research in which researchers develop and

characterize nanoclusters, manipulate their surface chemistry for

biological application, and demonstrate their ability to solve

biological problems. To integrate them for clinical diagnosis, the

toxicity [78] of fluorescent noble-metal nanoclusters to live cells

and even the whole body has to be dealt with more attention.

From our preliminary results [79], the labeling of endothelial

cells with fluorescent gold nanoclusters was found to intact

cellular functions including angiogenesis, vasodilatation,

coagulation, adhesion, and junction integrity. Human late

endothelial progenitor cells showed that the fluorescent gold

nanocluster-labeled cells not only exhibited strong fluorescence

but also possessed angiogenic potential in vitro and in vivo. But

some issues regarding how to remove these nanoclusters from

cells and animals should be studied in the future.

In order to advance in the field of nanobiotechnology, fully

investigating the fundamental properties of fluorescent

nanoclusters in biological systems is still required. Some recent

reports describing monofunctional [68,80-84] and

nanostructures assembly [68,85] techniques which give a key to

create future biosensors or nano machines such as artificial

viruses [86-89]. Combining the small fluorescent noble-metal

nanoclusters with other nanomaterials might assemble small but

complex multifunctional machines with dimensions similar to a

standard virus (< 150 nm). Additionally, multimodal imaging

probes using fluorescent noble-metal nanoclusters would

compensate many disadvantage of the one using organic dyes or

quantum dots. Multilevel imaging from molecular to medical

scales demonstrates the need for the development of advanced

nanoclusters that can be used in all kinds of imaging techniques,

such as PET, SPECT, MRI, CT, TEM and confocal microscopy

[90]. We conclude that the recent progress of fluorescent

noble-metal nanoclusters in synthesis and its biomedical

applications can offer new perspectives in fields ranging from

material chemistry, optical physics, cellular biology and medical

applications [91].

Acknowledgements

We gratefully acknowledge the support of research grants

from the Taiwan National Science Council (NSC

98-2627-B-033-001, 98-2627-B-195-001), Department of

Health Taiwan (DOH 98-TD-N-111-001), specific research

fields in Chung Yuan Christian University (CYCU-98-CR-BE),

and Mackay Memorial Hospital (MMH-E-98003). Dr. Lin was

supported by an NSC postdoctoral fellowship (NSC

098-2811-B-033-002).

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