the use of luminescent quantum

7/29/2019 The Use of Luminescent Quantum

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The use of luminescent quantum

dots for optical sensingJose M. Costa-Fernandez, Rosario Pereiro, Alfredo Sanz-MedelSemiconductor nanocrystals, known as quantum dots (QDs), have demon-

strated several remarkable, attractive optoelectronic characteristics espe-

cially suited to analytical applications in the (bio)chemical field. We review

progress in exploiting the attractive luminescent properties of QDs in

designing novel probes for chemical and biochemical optical sensing.

2005 Elsevier Ltd. All rights reserved.

Keywords: (Bio)chemical sensor; Nanostructure; Photoluminescence; Quantum dot

1. Introduction

Quantum dots (QDs) are nanostructured

materials [1], also known as zero-dimen-

sional materials, semiconductor nano-

crystals or nanocrystallites. These colloidal

nanocrystalline semiconductors, compris-

ing elements from the periodic groups

II-VI, III-V or IV-VI, are roughly spherical

and with sizes typically in the range 112

nanometer (nm) in diameter. At such

reduced sizes (close to or smaller than thedimensions of the exciton Bohr radius

within the corresponding bulk material),

these nanoparticles behave differently

from bulk solids due to quantum-

confinement effects [2,3]. Quantum

confinements are responsible for the

remarkable attractive optoelectronic

properties exhibited by QDs, including

their high emission quantum yields, size-

tunable emission profiles and narrow

spectral bands [3,4]. Moreover, their

strong size-dependent properties result in a

tunability emission that leads to new

applications in science and technology.

The past 20 years have seen intense

research activity in the fundamental study

of the synthesis and the photophysical

properties of QDs [58]. Different groups

have studied II-VI semiconductor QDs,

such as CdSe or CdS nanocrystals, in order

to characterize the relationship between

size, shape and electronic properties [2,4].

However, most applications so far have

focused on their use in microelectronics

and opto-electrochemistry (e.g., light-

emitting diodes, solar energy conversion

or quantum cascade lasers) [3,9,10].

The application of luminescent QDs as

biological labels was first reported in 1998

in two breakthrough papers [11,12]. Both

groups simultaneously demonstrated that

highly luminescent QDs can be madewater-soluble and biocompatible by

surface modification and bioconjugation.

They also showed the high potential of

QDs as highly sensitive fluorescent bio-

markers and (bio)chemical probes.

Other key advances enabling the

emerging practical applications of QDs in

biochemistry and medicine included the

synthesis of high-quality colloidal QDs in

large quantities [13] or recent advances

on surface chemistry of QDs by conjuga-

tion with appropriate functional molecules

[14]. The surface modification of QDs can

increase their luminescent quantum yields

[14], improve stability of the nanocrystals

and prevent them from aggregating [15],

and make QDs available for interactions

with target analytes [16], all of crucial

interest for chemical sensor or biosensor

applications.

This article deals with work on the

analytical applications of QDs in develop-

ing novel (bio)chemical sensors, an area of

growing interest in the past few years. We

include a brief discussion on the attractiveoptical properties of QDs and on the

importance of adequate control of the

synthesis and surface modification of

the luminescent QDs, in order to achieve

the desired selectivity and sensitivity for

sensing target analytes.

2. Optical properties

Studies of the physical properties of QDs

have revealed that strong confinement of

Jose M. Costa-Fernandez,

Rosario Pereiro,

Alfredo Sanz-Medel*

Department of Physical and

Analytical Chemistry,

University of Oviedo, c/ Julian

Clavera, 8, E-33006 Oviedo,

Spain

*Corresponding author.

Tel.: +34 985 10 34 74;

Fax: +34 985 10 31 25;

E-mail: [email protected]

Trends in Analytical Chemistry, Vol. 25, No. 3, 2006 Trends

0165-9936/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2005.07.008 2070165-9936/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2005.07.008 207
mailto:[email protected]:[email protected]


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excited electrons and holes in these nanocrystals exists

at such reduced sizes and led to observations of unique

optical and electronic properties [2,3]. These com-

pounds, which are usually non-fluorescing, develop an

intense, long-lasting luminescent emission when syn-

thesized on an nm scale. Semiconductor QDs are char-

acterized by a band-gap between their valence andconduction electron bands. When a photon having an

excitation energy exceeding the semiconductor band-gap

is absorbed by a QD, electrons are promoted from the

valence band to the high-energy conduction band. The

excited electron may then relax to its ground state by

the emission of another photon with energy equal to the

band-gap [4].

The results of quantum confinement are that the

electron and hole energy states within the nanocrystals

are discrete, but the electron and hole energy levels (and

therefore the band-gap) is a function of the QD diameter

as well as composition [17]. The band-gap of semicon-

ductor nanocrystals increases as their size decreases,resulting in shorter emission wavelengths [18,19]. This

effect is analogous to the quantum mechanical particle

in a box, in which the energy of the particle increases

as the size of the box decreases.

The size-dependent emission is probably the most

striking and the most studied optical property of QDs. As

the emission properties of semiconductor nanocrystals

depend strongly upon the energy and the density of the

electron states, they can be altered by engineering the

size and the shape of these tiny structures. For example,

Fig. 1 shows the fluorescence spectra of CdSe QDs with

different nanoparticle diameter sizes. As can be seen,

differently-sized CdSe nanocrystals can be tuned in the

500700-nm range. Moreover, as each material has

tunability limits, which depend on the physical limita-

tions of the dot size, other materials have been employed

in QD synthesis (see Table 1) (e.g., Zn-based QDs emitbelow 400 nm while Pb-based QDs have an emission in

the near-infrared spectral region).

As a result of their discrete, atom-like electronic

structure, QDs have typically very narrow emission

spectra with full width at half-maximum (FWHM) of the

luminescent emission of around 1540 nm. (QDs with

bandwidths as narrow as 12.716.9 nm FWHM have

been reported [20]). Since the emission lines are com-

paratively much narrower that those of organic dyes,

detection of the QDs suffers much less from cross-talk

that might result from the emission of a different fluo-

rophore bleeding into the detection channel of the fluo-

rophore of interest (analyte).On the other hand QDs typically exhibit higher fluo-

rescence quantum yields than conventional organic

fluorophores, allowing for greater analytical sensitivity.

The quantum yield of a luminophor is a function of the

relative influences of radiative recombination (producing

light) and non-radiative recombination mechanisms.

Non-radiative recombination, which largely occurs at

the nanocrystal surface, is a faster mechanism than

radiative recombination and is greatly influenced by the

surface chemistry. In this context, it has for example

5

0

0.2

0.4

0.6

0.8

1

500 550 600 650 700

Normalizedfluoresce

nce

intensity

Fluorescence

h(=400 nm)

3 nm~ 4 nm~ 7 nm~

155= 155= mnmn 095= 095= mnmn 746= 746= mnmn

Wavelength, nm

Figure 1. Size-tuneable fluorescence spectra of CdSe QDs. The diameter sizes of the nanoparticles are shown over the fluorescence spectrum.

Trends Trends in Analytical Chemistry, Vol. 25, No. 3, 2006

208 http://www.elsevier.com/locate/trac


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been demonstrated that capping the nanocrystal with a

shell of an inorganic wide-band semiconductor (e.g.,

ZnS) reduces such non-radiative deactivations and re-

sults in brighter emission [21]. Chan and Nie estimated

that single ZnS-capped CdSe QDs are about 20 times

brighter that single rhodamine 6G molecules [12].There is also evidence that QDs, suitably surface-

derivatized for protection, have also enhanced photolu-

minescent stability as compared to typical fluorescent

organic dyes. Several studies have demonstrated that the

photoluminescence properties of CdSe nanocrystals

(including the quantum yields, peak position and

FWHM) did not show any detectable change upon aging

in air for several months [22]. Moreover, QDs were ob-

served to be 100 times more stable that conventional

organic fluorophors against photobleaching [12].

3. Synthesis and surface chemistry

Progress on the synthesis of high-quality semiconductor

nanocrystals has played and is still playing a critical role

in the progress of QDs applications. Lithography-based

technologies have been widely used for QDs grown onto

adequate substrates [23], but have mainly been re-

stricted to the preparation of optoelectronic devices.

However, colloidal nanocrystals with single crystalline

structure and well-controlled size and size distribution

can be prepared by relatively simple nanocrystal-growth

processes, starting from organometallic precursors in a

mixed solvent [2,3], the latter approach being more

familiar to chemists.

Due to the availability of precursors and the simplicity

of crystallization, CdS and CdSe have been the most well-

studied colloidal QDs. Murray et al. [8] reported the

synthesis of high-quality Cd-chalcogenide nanocrystals

using dimethylcadmium as QD precursor in the presence

of a coordinating solvent at high temperatures. The most

common coordinating solvents used are trioctylphos-

phine oxide, trioctylphosphine and hexadecylamine

(frequently used together). Such solvents, which cap

the nanocrystal and stabilize its surface, determine the

particle solubility in organic media and prevent irre-

versible aggregation of the nanocrystals. However, QDs

capped with these hydrophobic coatings are incompati-

ble with aqueous assay conditions. Consequently, in

order to extend the field of application of the QDs,

hydrophilic capping agents must be introduced.A landmark in the development of wet chemical

routes for Cd-chalcogenide nanocrystals was the use of

thiols as stabilizing agents in aqueous solution [24].

Water-soluble nanoparticles were prepared by synthe-

sizing thiol-capped crystalline nanoparticles in aqueous

solution by using mercapto-alcohols (e.g., 2-mercap-

toethanol or 1-thioglicerol) and mercapto-acids (e.g.,

thioglycolic acid or thiolactic acid) as stabilizers [24].

In an important paper [13], Pengs group reported the

synthesis of high-quality CdTe, CdSe and CdS nano-

crystals using CdO as precursor instead of Cd(CH3)2. This

latter compound is toxic, unstable, explosive andexpensive, rendering QD-synthesis schemes based on its

use unsuitable for large-scale synthesis (due to the need

of critical experimental conditions). The quality of the

QDs synthesized with this new approach [13] was found

to be comparable or superior to the best previously re-

ported. Moreover, the reported synthesis scheme (see

Fig. 2) proved to be reproducible, were based on mild and

simple conditions, and had great potential to be scaled

up for industrial applications.

In recent years, other alternative routes for synthesis of

highly mono-dispersed QDs have been investigated. For

example, the use of stable non-air-sensitive precursors

based on selenocarbamate derivatives of Zn or Cd [25] or

on the air-stable complex Cd imino-bis(diisopropylphos-

phine selenide) [26] have been proposed to synthesize

monodispersed luminescent QDs of comparable quality to

those prepared by more conventional methods.

However, to ensure efficient emission, any traps for

the photogenerated electron and hole should be avoided.

Possible traps in QDs are generally surface atoms that

are missing at least one chemical bond. The surface

atoms must be optimally constructed or reconstructed

and passivated with some ligands to get rid of traps.

Coating nanoparticles with a different semiconducting

Table 1. Nanocrystal materials and range of tunability

QD core materiala Fluorescence-emission range (nm) QD-diameter range (nm)

Zinc sulfide (ZnS) 300410 Zinc selenide (ZnSe) 370430

Cadmium sulfide (CdS) 355490 1.96.7

Cadmium telluride (CdTe) 620710

Lead sulfide (PbS) 700950 2.39Lead selenide (PbSe) 12002340Lead telluride (PbTe) 18002500

aEmission fluorescence of core-shell QDs is also within the range given in the second column.


http://www.elsevier.com/locate/trac 209


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material was shown to have a profound impact on the

photophysics of the nanocrystalline core [2,14,21].

Deposition of a semiconductor layer with a large band-

gap (Eg) relative to the core typically results in the

enhancement of the QD emission due to the suppressionof radiationless recombination mediated by surface states

[2,21], while the degree of charge-carrier confinement

does not change. Conversely, an outer layer from a

semiconductor with a small Eg provides an additional

area of delocalization for electron and hole [2,14,21]. Of

course, relaxation of the confinement regime results in a

red shift of the spectral features.

The exciting size-dependent and surface-dependent

properties of nm-sized QDs have stimulated research on

surface modification of QDs, aiming to expand their

practical applications. In this context, the conjugation of

a semiconductor nanoparticle with an organic molecule

[27], able to interact selectively with a target molecule or

(bio)chemical species, extends the area of applications

from the electronic or optical devices to the biological or

chemical systems, such as the preparation of non-

radioactive biological labels [11,12] or chemical opto-

sensors [16].

4. Optical sensing with quantum dots

More than five years have elapsed since QDs were first

proposed as stable luminescent probes in biological

labeling applications. In that pioneer work, Alivisatoss

group [11] reported a link between biomolecules and

CdS or ZnS core-shell CdSe QDs via surface coating with

an additional layer of silica in order to make them bio-

compatible and water soluble, and established the utilityof the nanocrystals for biological staining. Simulta-

neously, Chan and Nie [12] linked biomolecules to

water-soluble and biocompatible QDs surface-modified

with mercaptoacetic acid for ultrasensitive detection at

the single-dot level. They demonstrated that conjuga-

tion of the QDs with appropriate immunomolecules

can be used for recognition of specific antibodies or

antigens by measuring the luminescence emission of the

nanoparticles.

Many authors have stressed the distinct advantages of

QD bioconjugates over conventional organic dyes (such

as rhodamine), namely greater brightness, greater sta-

bility with respect to photobleaching and narrower

spectral line-widths. However, QD biological labeling has

been slow to emerge into common practice, partly due to

the difficulty in producing stable QD-biomolecule com-

plexes. Developments have stressed the importance of

adequate surface modifications in developing lumines-

cent QDs for labeling in bioanalysis and diagnostics, as

tags for protein and DNA immunoassays or as biocom-

patible labels for in vivo imaging studies. Several reviews

have summarized the use of luminescent QDs in such

biochemical applications [2731]. Moreover, the fast

development and improvements in the synthesis of QDs

(a) NANOPARTICLE SYNTHESIS

(b) SURFACE MODIFICATION

-S-CH2-COO(-)

QD

Water-soluble QDs

TOP/TOPOCdSe QDs

(in CH4)

Reflux 12 h.

Thermom

eter syrin

ge

H2HS-C-COOH

Purification

QDs separation(at~ 15,000 rpm)

andre-dispersion in H2O

P

P

P

O

P

OQD

P

O

P TOP

TOPO

TOPO+

CdO+

HPA

TOP-Se

320 C

Thermom

eter syringe

Argon

Nucleation GrowthQD

20 C270 C

Argon

~ 20 min

Purification

QDs separation(at~ 15,000 rpm)

andre-dispersion in CH4

P

P

P

O

P

OQD

Dilution with~ 10 mL CHCl3

Figure 2. Schematic illustration of a typical synthesis process and surface-modification of a luminescent QD based on the use of CdO asprecursor. TOP: trioctylphosphine; TOPO: trioctylphosphine oxide; HPA: Hexylphosphonic acid.




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have uncovered possibilities that analytical chemists

have also started to explore in developing these

nanomaterials for a new generation of optical sensors

based on luminescence.

4.1. Fluorescence-based transduction

As the luminescence of QDs is very sensitive to the sur-face states of the QDs, it is reasonable to expect that the

chemical or physical interactions between a given

chemical species and the surface of the nanoparticles

would result in changes in the efficiency of the core

electron-hole recombination [32]. This has been the

basis of the increase in research activity on the devel-

opment of novel optical sensors based on QD probes.

Following this approach, Cd-based QDs have been re-

ported for optical sensing of small molecules and ions

(Table 2). In a pioneering work, the addition of Cd ions

to a basic aqueous solution containing unpassivated CdS

nanoparticles resulted in important enhancement of the

luminescence quantum yield of the nanoparticles,without detectable changes in particle sizes [7]. This

effect was attributed to the formation of a Cd(OH)2 shell

on the CdS core, which effectively eliminates the non-

radiative recombination of charge carriers.

A similar photoluminescence-activation effect (attrib-

uted to passivation of surface trap sites that are either

being filled or energetically moved closer to the band

edges by this simple chemical process) was also induced

after adding Zn and Mn ions to colloidal solutions of CdS

or ZnS QDs [32,33]. This behavior provided the basis for

optical sensing of such metallic cations with QDs.

Besides the activation effect, QD-based optical sensingquenching strategies (based on the quenching by the

analyte that affects the luminescence emission of the

nanoparticle) have been proposed. Quenching mecha-

nisms to explain how metal ions quench fluorescence

of QDs include inner filter effects, non-radiative recom-

bination pathways, electron-transfer processes and

ion-binding interactions. Measurement of the lumines-

cence-deactivation ratio of peptide-coated CdS QDs has

been proposed for the optical sensing of Cu(II) and Ag(I)

[34]. Similarly, the effect of three different ligands

(L-cysteine, thioglycerol and polyphosphate) was evalu-

ated on the luminescence deactivation of water-soluble

CdS QDs with respect to several cations, including Zn

and Cu ions [35]. This latter work was one of the first

references to the use of luminescent QDs as selective ion

probes in aqueous samples.

Isarov and Chrysochoos [36] observed that the addi-

tion of Cu(II) perchlorate in 2-propanol to CdS nano-

particles led to the binding of copper ions onto the QD

surface, accompanied by rapid reduction of Cu2+ to Cu+.

It was proposed that copper ions bound onto the surface

of the QDs facilitate non-radiative electron/hole (e/h+)

annihilation, thus resulting in a quenching of the

luminescence from the nanoparticles. It was shown thatTable2.

QD-basedfluorescentprobes

forchemicaldeterminationofsmallmoleculesandions

QD

material

QD

coating

Analyte

Matrix

Detectionlimit

Measuringsignal

Ref.

CdS

Cly-His-Leu-Leu-Cys

Cu(II)

Phosphatebuffer

0.5lM

Fluorescencequenching

[34]

Ag(II)

CdS

Polyphosphate

Cu(II)

Water

0.8mMZn(II)


[35]

L-cysteine

Fe(III)

0.1mMCu(II)

Thioglycerol

Zn(II)

CdSe

2-mercaptoethanesulfonicacid

Cu(II)

Water

3.2nM


[37]

CdSe-ZnS

Bovineserumalbumin

Cu(II)

Water

10n

M


[38]

CdSe

Mercaptoaceticacid+bovineserumalbumin

Ag(I)

Water

70n

M


[39]

CdTe

3-mercaptopropionic

acid

Cu(II)

Water

0.19

ng/mL


[40]

CdTe

Thioglycolicacid

Zn(II),

Mn(II),

Ni(II),Co(II)

Water

Fluorescencequenching-en

hancement

[41]

CdS

Polyphosphate

I

Methanol


[42]

CdSe

Tert-butyl-n-(2-mercaptoethyl)-carbamate

CN

Methanol

0.1lM


[44]

CdSe

2-mercaptoethanesulfonicacid

CN

Water

1.1lM


[45]

CdS

L-cysteine

Ag

+

Water

5.0nM

Fluorescenceenhancement

[46]

CdSe

Incorporatedinpolym

erfilms

Triethylamine

Gasmedia

Fluorescencequenching-en

hancement

[47]

Benzylamine

CdSe-ZnS

Thioglycolic+organo

phosphoroushydrolase

Paraoxon

Water

10n

M


[49]




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the quenching could be employed for chemical sensing of

Cu ions in the organic solution.

Water-soluble CdSe QDs with their surface modified

with 2-mercaptoethane sulfonic acid can be used for the

sensitive and selective determination of copper (II) ions

in aqueous solutions, based on fluorescence-quenching

measurements [37].In addition, based on the photoluminescence

quenching of the nanocrystals, CdSe-ZnS QDs modified

with bovine serum albumin (BSA) were investigated for

the determination of copper [38], CdSe QDs modified

with mercaptoacetic acid and BSA were assayed for the

analysis of silver [39] and CdTe nanocrystals modified

with mercaptopropionic acid were proposed for the

determination of Cu(II) ions [40]. It was observed that

the change in the absorption spectra caused by Cu(II)

can be reversed by the addition of EDTA, a good com-

plexing agent for Cu(II) ions. Thus, the authors proposed

that the interaction between Cu(II) ions and the QD

surface should be of the ion-binding type.Li et al. [41] synthesized water-soluble luminescent

thiol-capped CdTe QDs and investigated the effect of

divalent metal ions on their photoluminescence re-

sponses. They found that zinc ions enhanced the lumi-

nescence emission of the QDs. However, other metals

(e.g., calcium, magnesium, manganese, nickel and cad-

mium) quenched luminescence.

Apart from research on QD-based fluorescent sensors

for ion metals, work on other chemical species (e.g., io-

dide [42] or cyanide [43]) has reported quenching the

emission of CdS or CdSe QDs. A polyphosphate-stabilized

CdS QD was evaluated for optical sensing of iodide [42]and found strong decay of luminescence intensity (decay

times 10 ls), brought about by the analyte. Such

quenching effects [42] were attributed to inner filter

effects, non-radiative recombination pathways and

electron-transfer processes.

The strong, reversible adsorption of negatively-

charged CN onto the QD surface, with the consequent

increased location due to compression of the electron-

wave function in the QDs, was used to explain the

quenching effect of cyanide [43].

Following this mechanism, the synthesis of red

photoluminescent CdSe QDs, with their surface modified

with tert-butyl-n-(2-mercaptoethyl)-carbamate, has

been proposed for the selective, sensitive determinationof free cyanide in methanol after a photoactivation of the

QDs [44].

In a further work [45], the authors reported the syn-

thesis of water-soluble luminescent CdSe QDs, surface-

modified with 2-mercaptoethane sulfonate, for the

selective determination of free cyanide in aqueous solu-

tion (see Fig. 3). The addition of surfactant agents to the

measurement aqueous solution was found to further

greatly stabilize the QDs. In this way, the fluorescent

signals observed allowed for high sensitivity (detection

limit 1.1 106 M) and also for great selectivity of the

proposed cyanide detection (over many other anionic

species).As can be seen, most of the methods described so far

rely on the chemical sensing of small molecules and ions

with QDs via analyte-induced deactivation of photolu-

minescence. However, Zhu and Chen [46] proposed a

method for the determination of trace levels of silver ion

based on luminescence enhancement of water-soluble

CdS QDs modified with L-cysteine. The authors showed

detection limits as low as 5.0 109 M. They proposed

that the fluorescence-enhancement effect could be

attributed to the formation of a complex between silver

ions and the RS- groups adsorbed on the surface of the

modified QDs, which resulted in the creation of radiativecenters at the CdS/Ag-SR complex.

The interactions between some reactive gas molecules

and the surface of CdSe QDs have also been exploited in

developing gas-sensing technologies [47]. Nazzal et al.

found that the photoluminescence of CdSe QDs incor-

porated into polymer thin films is reversibly enhanced

or quenched by the presence of certain gases in the

0

100

200

300

450 550 650

Wavelength, nm

IF

0

100

200

300

450 550 650

Wavelength, nm

IF

+ CN-

CdSeCdSeCdSeCdSeCdSe CdSeCdSeCdSeCdSeCdSeC N-

C N-

C N-

Figure 3. Effect of the addition of 0.65 mg/l of cyanide to the luminescence emission spectra of CdSe QDs surface-modified with MES.




7/12

environment. After QD synthesis, photostimulation was

found to be necessary to obtain a stabilized emission

profile and to provide reliable responses to the presence

of the gases. This effect, shown in Fig. 4, was also re-

ported by other groups [37,44,45]. Although the

mechanism(s) explaining this photoactivation is (are)

not clear, it is thought that a reconstruction of the sur-face atoms of the nanoparticle, or an optimization of

surface-ligand passivation, could lead to the observed

enhancement of the measured luminescence [47].

QDs were also proposed for the design of sensing

assemblies for selective detection of paraoxon [48].

Water-soluble CdSe QDs, surface functionalized with

thioglycolic acid, were synthesized and incorporated to-

gether with organophosphorous hydrolase (OPH) in a

thin film prepared by the layer-by-layer technique. The

presence of paraoxon in the sample solution was de-

tected by changes in the photoluminescence emission of

the QDs, attributed to an interaction of the analyte with

the OPH included in the sensing film, changing itsconformation.

Following this approach, the synthesis of (CdSe)ZnS

nanocrystals and their conjugation with organophos-

phorous hydrolase (through electrostatic interaction

between negatively-charged QD surfaces and the posi-

tively-charged protein side chain and -NH2 ending

groups) has been proposed in developing a biosensor to

detect paraoxon, obtaining detection limits as low as

108 M [49]. The photoluminescence intensity of the

OPH/QD bioconjugate was quenched in the presence of

paraoxon, matching very well with the Michaelis-

Menten equation. This result indicated that the quenching

was caused by the conformational change in the en-

zyme, which was confirmed by gas-chromatography

measurements. Although such a strategy of QD-surfacebioconjugation has not yet been exploited frequently for

sensing, it holds great potential for further developments

in optical sensing with QDs.

In recent years, QDs have been used as inorganic,

non-specific, DNA-binding proteins that act as lumines-

cent labels for different applications (e.g., multicolor gene

mapping on the nm scale) [50]. Moreover, fluorescence

quenching of water-soluble CdSe QDs, surface modified

with mercaptoacetic acid, has also been used to develop

a fluorescence probe for rapid, sensitive determination of

DNA in a neutral medium [51]. The mechanism for the

binding of the nucleic acids to the QDs was investigated

and it was concluded that nanoparticles bind to the helixstructure of the DNA in a non-intercalative way,

resulting in the observed deactivation of the lumines-

cence emission of the QDs.

4.2. Fluorescence (or Forster) resonance

energy-transfer-based sensors

Energy-transfer mechanisms have been widely used in

different fields and are the basis of a new generation of

Figure 4. Effect of photoactivation (a) fluorescence spectra from CdSe quantum dots measured after different sunlight time exposures (from ref-erence [44]), (b) Pictures of a methanolic QDs solution (1) freshly prepared and (2) after 3 days exposed to the sunlight.




8/12

luminescent sensors [52]. In this context, the capability

of tailoring (via size) QD-photoemission properties should

allow efficient energy transfer with a number of con-

ventional organic dyes, thus suggesting the use of the

nanoparticles in sensor or chemical assay applications

based on photochemically-induced fluorescence (or

Forster) resonance-energy-transfer (FRET) mechanisms.However, the QD emission spectrum is narrower and

more symmetric than the emission from conventional

organic fluorophores, making it much easier to distin-

guish the emission of the donor from that of the accep-

tor. Moreover, the high quantum yields of QDs make

energy transfer very efficient.

Several studies have already confirmed that QDs are

excellent candidates for use in the design of novel FRET-

based strategies. As an example, specific binding of dif-

ferent proteins was observed via measurements of FRET

between a CdSe-ZnS QD donor, attached to one of the

proteins, and some organic acceptor dyes attached to the

other protein under study. In the presence of specificinteractions between both proteins, strong enhancement

of the acceptor-dye fluorescence was observed [53].

In a more fundamental study, conjugation of BSA

with luminescent CdTe nanoparticles (capped with L-

cysteine) resulted in a significant increase in the CdTe

fluorescent emission, attributed to an efficient reso-

nance-energy transfer from the tryptophan moieties of

the protein units to the CdTe nanoparticles acting as

acceptors [54]. However, despite the demonstrated

favourable properties of luminescent QDs for FRET

experiments, only very few studies on the synthesis of

QD bioconjugates and their applications for QD-FRET-based optical sensors have been published so far.

Luminescent CdSe QDs have been used as energy do-

nors in developing a competitive FRET assay for maltose

[55] (see Fig. 5). Semiconductor nanoparticles biocon-

jugated to different maltose-binding proteins, formed

using a non-covalent self-assembly scheme, act as the

resonance-energy-transfer donors, while non-fluorescent

dyes bound to cyclodextrin serve as the energy-transfer

acceptors. In the absence of maltose, cyclodextrin-dye

complexes occupy the protein binding sites. Energy

transfer from the QDs to the dyes quenches the QD

fluorescence. When maltose is present, it replaces the

cyclodextrin complexes, and the QD fluorescence recov-

ers [55]. This approach has been successfully employed

in developing a prototype QD-based sensor for sensing

maltose in solution [56].

CdSe-ZnS core-shell biocompatible QDs, stabilized by

mercaptopropionic acid modified with a thiolated oligo-

nucleotide, have been proposed as energy donors for

lighting up the dynamics of telomerization or of DNA

replication occurring on the nanoparticles, using FRET

to dye units incorporated into the new synthesized

telomere or DNA replica [57]. After addition of telome-

rase, during the progression of the telomerization, the

fluorescence emission from the QDs at 560 nm decreaseswith the concomitant increase of the 610 nm emission of

the dye (using 400-nm excitation). Emission observed

upon telomerization is attributed by the authors to FRET

from the QDs to the dye molecules incorporated into the

telomeric units by telomerase. The CdSe-ZnS QDs func-

tionalized with M 13~ DNA also enabled the detection of

a viral DNA by following the DNA-replication process by

FRET. Results can be applied to the fast, sensitive

detection of cancer cells [57]. It could be also applied to

the development of chip-based DNA sensors as it func-

tions like logic gates, where FRET readout occurs when

hybridization and replication proceed.

In one application, luminescent ZnS-capped CdSe QDs,

covered with mercaptoacetic acid, have been conjugated

to amine-terminated molecular beacons (MBs) at the 5 0

end for probing DNA sequences [58]. Connected to the 3 0

end of the molecular beacon, there is a quencher mole-

cule [4-(40-dimethylaminophenylazo) benzoic acid,

DABCYL]. In the absence of the target DNA sequence,

MBs form a hairpin structure in which the QDs and

DABCYL are in such close proximity that energy from

the QDs is transferred to the quencher and no fluorescent

signal is observed. After adding the target DNA se-

quence, the MB structure opens. Since QD and quencher

DQ PBM

excitation(350nm)

FRET Quenching

Dye

DC

DQ PBM

excitation(350nm)

maltose+

ecnecseroulF

Dye

DC

a

b

Figure 5. Schematic diagram of the quantum-dot based FRET malt-ose sensor (adapted fromreference [56]). QDs conjugated to around10 maltose-binding proteins function as the FRET donors. Non-fluorescent dyes bound to a cyclodextrin serve as the acceptorsand in the absence of maltose are filling the protein binding sitesresulting in a quenching of the luminescence. Whenmaltose is pres-ent, it removes the cyclodextrin-dye complex and the fluorescenceis recovered. MBP: maltose binding protein, CD: cyclodextrin.




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are then separated from each other, no FRET occurs and

QD emission can be detected. The authors demonstrated

that using QDs in this probe resulted in an improved

lifetime during imaging, as compared to using organic

fluorophores.

The application of water-soluble ZnS nanoparticles,

surface-modified with sodium thioglycolate, as fluores-cence probes has been described for specific determination

of protein content in a serum sample (e.g., human serum

albumin, BSA and gamma globulin) with detection limits

of the order of 10 pg/mL [59]. Energy transfer from sur-

face-adsorbed proteins to the nanoparticles has been

proposed as the mechanism responsible of enhancing the

QD luminescence used for sensing. The methodology was

applied to the analysis of human serum samples, and re-

sults obtained were in good agreement with those given by

alternative, conventional techniques.

The use of luminescent QDs conjugated to appropriate

antibody fragments has been employed to develop solu-

tion-phase, nanoscale, sensing assemblies for detectingthe explosive 2,4,6-trinitrotoluene (TNT) in aqueous

environments based on FRET measurements [60]. The

presence of TNT was detected by displacing the dye-

labeled analogue bonded to the QD surface, resulting in

the elimination of FRET and in the concentration-

dependent recovery of QD photoluminescence.

It should be mentioned that QD-FRET assays can be

designed so that FRET is the dominant energy-transfer

process, but FRET efficiency is still inherently low com-

pared to that of conventional dyes (due to the compar-

atively large size of QDs, it is too difficult to secure close

enough proximity for FRET to occur efficiently). How-ever, several studies have been carried out in order to

gain a better understanding of the process, showing that

enhanced efficiency can be obtained by careful design of

the QD-bioconjugation scheme. Using the FRET scheme

for maltose determination [55], mentioned previously,

the authors found that, by attaching several active dye-

labeled proteins to the QD surface, the overall FRET

signal was improved substantially over a simple one

donor-to-one acceptor FRET pair [61]. Efficiency was

further enhanced by increasing the number of dye

acceptors in the QD bioconjugate, where QDs functioned

as efficient energy donors [61]. Furthermore, the large

size of QD fluorophores, compared to organic dyes,

allowed design of configurations where, for example,

multiple acceptors could coordinate around and interact

with a single QD donor. This strategy, already demon-

strated for multiple detection in immunoassays [62],

suggests the possibility of achieving mutianalyte opto-

sensing using a single QD donor and multiple acceptors

in a FRET-assay format.

4.3. Surface-plasmon-resonance applications

QDs have been also investigated by measuring surface-

plasmon resonance (SPR). Redox transformations

occurring on chemically-modified surfaces may signifi-

cantly alter the refractive index of the interface and thus

induce changes in the plasmon angle of the SPR spectra

[63]. This approach was followed in the design of an SPR

sensor for acetylcholine-esterase inhibitors based on the

photoelectrochemical-charging effect of Au nanoparti-

cles in an Au-nanoparticle/CdS-QD array (coating anAu/glass surface), which was followed by means of SPR

changes upon continuous irradiation of the sample [63].

The fact that other enzymes may be coupled to the Au-

semiconductor-nanoparticle array and so activate

photoelectrochemical functions suggests that using SPR

spectroscopy combined with surface-modified QDs could

provide an alternative tool for new SPR (bio)sensor

probes.

4.4. Phosphorescence transduction

Only fluorescence transduction has so far been employed

for photoluminescence sensing in combination with QDs.

However, investigation of the luminescence properties ofQDs is slowly expanding into phosphorescence, a detec-

tion principle that may provide several advantages for

the design of reliable optical sensors [64,65].

The dopage of sol-gel porous matrices with Tb2S3 QDs

has been found to produce photoluminescent materials

with an emission comprising two well-defined bands,

one at 440 nm (that corresponds to the undoped sol-gel)

and the other at 600 nm that the authors attributed to

the Tb2S3 nanoparticles in the silica xerogel [66]. This

last emission presents characteristics typical of a room-

temperature phosphorescence (RTP) emission, although

the origin of the luminescence and the emission mech-anism is not yet understood [66].

Moore et al. from Mercer University have also reported

phosphorescence emission from aqueous mixed sulfide

QD matrices, QD-CdxZn1-xS, doped with manganese(II)

[67]. The authors evaluated the impact of matrix com-

position on the QD phosphorescence ($590 nm). They

found that the observed RTP intensity for the CdxZn1-

xS:Mn QDs was very sensitive to matrix composition

(e.g., the 590 nm emission increased with the Zn con-

centration of the matrix).

Although very preliminary, those studies seem to open

the door to novel transduction schemes and applications

of QDs for optical sensing.

4.5. Immobilization techniques

Most of the work on QD applications so far has been

restricted to solution-sensing assays. A step further to-

wards developing useful optosensing approaches [68]

consists of immobilizing those QDs in appropriate solid

supports to fabricate active solid phases for working in

flowing solutions [65].

In this context, sol-gel materials have been demon-

strated to be especially suited to the development of

luminescent optical sensors by trapping the indicator




10/12

molecules inside the inorganic structure during the

polymerization process [69]. Several approaches have

been also proposed for synthesis of sol-gel materials

doped with QDs [7072]. Most of the synthetic routes

involve preparing and surface modifying the QDs in

solution followed by sol-gel processing in order to obtain

an inorganic material doped with the luminescentnanocrystals. Considering that QDs are very sensitive to

changes in the environment, the transfer of these

materials into glasses, through sol-gel processes, is not a

simple task. Changes in solvent polarity or QD surface

reactions during sol-gel polymerization would result in

an undesirable quenching of the QD photoluminescence.

In order to overcome such limitations, several ap-

proaches have been investigated, including the use of

alkyl amines as a bifunctional aid in QD-glass synthesis

(amines act as gelation catalysts and stabilizers) [70].

Such QD glasses have demonstrated high stabilities

and resistance to degradation, so they have been mainly

used for optoelectronic applications (e.g., solar concen-trators or as active media in tunable lasers [73]). How-

ever, these sol-gel materials, doped with luminescent

QDs, are also expected to be also for optosensing appli-

cations in the near future.

A related approach is to incorporate QDs into

molecularly imprinted polymers (MIPs) [74], which act

as artificial receptors/antibodies exhibiting tailor-made

selectivity for a given template molecule. Following

this approach, Lin et al. [75] synthesized different

MIPs with several templates incorporating CdSe/ZnS

core-shell QDs, derivatized with 4-vinylpiridine. Adding

the functionalized QDs to the monomers, cross-linkersand template molecules in the precursor mixture

incorporated the nanocrystals into the MIP during

polymerization. Optosensing of the analytes is achieved

by measuring the quenching of the photoluminescent

emission from the QDs included in the polymeric

structure. Such quenching is attributed to fluores-

cence-energy-transfer processes between the QDs and

the template molecules. The approach has been suc-

cessfully tested for caffeine detection, although addi-

tional work needs to be carried out to characterize this

optosensor analytically. It is clear that this approach

also opens up a new avenue for the development of

new QD-based optical sensors.

The advantages and the disadvantages of using the

different optical transduction strategies already at-

tempted in developing chemical sensors based on QDs

can be summarized as follows:

i Methods based on chemical or physical interactions

between target chemical species and the surface of

the nanoparticles are very simple, easy to develop

and have demonstrated very high sensitivity and

selectivity features. However, those methods appear

to be restricted to sensing just a few reactive small

molecules or ions.

ii Quantum dots have been demonstrated to be espe-

cially suited to the development of new chemical

sensors based on energy-transfer phenomena. This

approach will probably be widely used as a general

strategy to develop new QD-based sensor systems

for analytes unsuitable for direct analysis via interac-

tion with QD particles. Of course, those methods arenot as simple as those in i) because many different

parameters need to be carefully controlled (e.g.,

distance of the acceptor/donor dyes to the QD sur-

face, and orientation and number of groups) in order

to achieve an analytically useful energy-transfer

process.

iii RTP methods offer general, exceptional characteristics

in terms of sensitivity and selectivity and some other

advantages over fluorescence methods. However,

work on the development of chemical sensors based

on QDs using phosphorescence transduction is still

at its very early preliminary development stages.

Thus, the practical usefulness of RTP methods hasnot yet been demonstrated.

5. Conclusions and future prospects

The popularity of QDs as photoluminescent probes

for optical sensing is steadily increasing, as research-

ers move to exploit the unique properties of this new

class of luminophores. Optosensing technologies will

probably combine the important advantages of QDs

with flow-analysis techniques and perhaps fibre-opticinstrumentation.

Chemical-sensing developments will benefit from the

continuous advances taking place in the science of

QDs. Thus, future improvements in the nature, range

and quality of prepared nanomaterials can be ex-

pected. Chemical-surface modifications of the QDs

have still to be perfected in order to enhance the

selectivity of the systems and to profit from their

favorable emission features. In this context, the con-

jugation of selective reagents to the surface of lumi-

nescent QDs (a strategy well established for imaging,

immunoassay and labeling applications in biological

science) appears to be a most promising strategy in

further developing bioactive fluorescent probes for

sensing applications.

Moreover, approaches such as the combination of the

nanoparticles with energy-transfer processes, phospho-

rescence detection or inclusion on MIPs are promising

possibilities that are now being investigated.

Last, but not least, QDs should now be integrated into

appropriate solid supports, a process that has only just

begun, in order to develop reliable active phases and

optosensors able to provide useful flow-through optical

sensing or fiber-optic-based sensing applications.




11/12

In brief, the future of QDs for optical sensing looks

bright, as their analytical potential in the field now starts

to be realized.

Acknowledgement

Financial support from the EU Project SWIFT-WFD

(Contract SSPI-CT-2003-502492) and MAT2003-

09074-C02 (Feder Programme and Ministerio de Ciencia

y Tecnologa, Spain) is gratefully acknowledged.

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