You are on page 1of 12

Trends in Analytical Chemistry, Vol. 25, No.

3, 2006

Trends

The use of luminescent quantum dots for optical sensing


M. Costa-Ferna ndez, Rosario Pereiro, Alfredo Sanz-Medel Jose
Semiconductor nanocrystals, known as quantum dots (QDs), have demonstrated several remarkable, attractive optoelectronic characteristics especially suited to analytical applications in the (bio)chemical eld. 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
M. Costa-Ferna ndez, Jose Rosario Pereiro, Alfredo Sanz-Medel* Department of Physical and Analytical Chemistry, n University of Oviedo, c/ Julia a, 8, E-33006 Oviedo, Claver Spain

Corresponding author. Tel.: +34 985 10 34 74; Fax: +34 985 10 31 25; E-mail: asm@uniovi.es

Quantum dots (QDs) are nanostructured materials [1], also known as zero-dimensional materials, semiconductor nanocrystals or nanocrystallites. These colloidal nanocrystalline semiconductors, comprising 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 the dimensions of the exciton Bohr radius within the corresponding bulk material), these nanoparticles behave differently from bulk solids due to quantumconnement effects [2,3]. Quantum connements are responsible for the remarkable attractive optoelectronic properties exhibited by QDs, including their high emission quantum yields, sizetunable emission proles 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., lightemitting diodes, solar energy conversion or quantum cascade lasers) [3,9,10]. The application of luminescent QDs as biological labels was rst reported in 1998 in two breakthrough papers [11,12]. Both groups simultaneously demonstrated that highly luminescent QDs can be made water-soluble and biocompatible by surface modication and bioconjugation. They also showed the high potential of QDs as highly sensitive uorescent biomarkers 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 conjugation with appropriate functional molecules [14]. The surface modication 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 developing novel (bio)chemical sensors, an area of growing interest in the past few years. We include a brief discussion on the attractive optical properties of QDs and on the importance of adequate control of the synthesis and surface modication 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 connement of 207

0165-9936/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2005.07.008

Trends

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

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 compounds, which are usually non-uorescing, develop an intense, long-lasting luminescent emission when synthesized on an nm scale. Semiconductor QDs are characterized by a band-gap between their valence and conduction 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 connement 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 semiconductor 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 uorescence 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 limitations of the dot size, other materials have been employed in QD synthesis (see Table 1) (e.g., Zn-based QDs emit below 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 comparatively 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 uorophore bleeding into the detection channel of the uorophore of interest (analyte). On the other hand QDs typically exhibit higher uorescence quantum yields than conventional organic uorophores, allowing for greater analytical sensitivity. The quantum yield of a luminophor is a function of the relative inuences 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 inuenced by the surface chemistry. In this context, it has for example

Fluorescence
=551 n nm m nm m =590 n = 647 n nm m

h( =400 nm)

~ 3 nm
1

~ 4 nm

~ 7 nm

Normalized fluorescence intensity

0.8 0.6 0.4 0.2 0 500 5 550 600 650 700

Wavelength, nm
Figure 1. Size-tuneable uorescence spectra of CdSe QDs. The diameter sizes of the nanoparticles are shown over the uorescence spectrum.

208

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

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

Trends

Table 1. Nanocrystal materials and range of tunability QD core materiala Zinc sulde (ZnS) Zinc selenide (ZnSe) Cadmium sulde (CdS) Cadmium telluride (CdTe) Lead sulde (PbS) Lead selenide (PbSe) Lead telluride (PbTe)
a

Fluorescence-emission range (nm) 300410 370430 355490 620710 700950 12002340 18002500

QD-diameter range (nm)

1.96.7

2.39

Emission uorescence of core-shell QDs is also within the range given in the second column.

been demonstrated that capping the nanocrystal with a shell of an inorganic wide-band semiconductor (e.g., ZnS) reduces such non-radiative deactivations and results 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 surfacederivatized for protection, have also enhanced photoluminescent stability as compared to typical uorescent 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 observed to be 100 times more stable that conventional organic uorophors 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 restricted 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 wellstudied 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 trioctylphosphine 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 irreversible aggregation of the nanocrystals. However, QDs capped with these hydrophobic coatings are incompatible with aqueous assay conditions. Consequently, in order to extend the eld 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 synthesizing thiol-capped crystalline nanoparticles in aqueous solution by using mercapto-alcohols (e.g., 2-mercaptoethanol 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 nanocrystals using CdO as precursor instead of Cd(CH3)2. This latter compound is toxic, unstable, explosive and expensive, 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 reported. 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(diisopropylphosphine selenide) [26] have been proposed to synthesize monodispersed luminescent QDs of comparable quality to those prepared by more conventional methods. However, to ensure efcient 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 209

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

Trends

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

(a) NANOPARTICLE SYNTHESIS


m er Th er et om

sy rin ge

Argon

TOP-Se

Argon

Dilution with ~ 10 mL CHCl3

P O

TOPO

TOPO + CdO + HPA 320 C


Nucleation

TOP

270 C
Growth

20 C
Purification
P O

QD
~ 20 min QDs separation (at~ 15,000 rpm) and re-dispersion in CH4

(b) SURFACE MODIFICATION


H2 HS-C-COOH
sy rin ge
m er Th er et om

P P O O

QD
P

TOP/TOPO CdSe QDs (in CH4)

Purification
QDs separation (at~ 15,000 rpm) and re-dispersion in H2O

QD
-S-CH2-COO(-)

Reflux 12 h.

Water-soluble QDs
Figure 2. Schematic illustration of a typical synthesis process and surface-modication of a luminescent QD based on the use of CdO as precursor. TOP: trioctylphosphine; TOPO: trioctylphosphine oxide; HPA: Hexylphosphonic acid.

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 bandgap (Eg) relative to the core typically results in the enhancement of the QD emission due to the suppression of radiationless recombination mediated by surface states [2,21], while the degree of charge-carrier connement 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 connement 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 modication 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 nonradioactive biological labels [11,12] or chemical optosensors [16].

4. Optical sensing with quantum dots More than ve years have elapsed since QDs were rst proposed as stable luminescent probes in biological 210

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 biocompatible and water soluble, and established the utility of the nanocrystals for biological staining. Simultaneously, Chan and Nie [12] linked biomolecules to water-soluble and biocompatible QDs surface-modied with mercaptoacetic acid for ultrasensitive detection at the single-dot level. They demonstrated that conjugation of the QDs with appropriate immunomolecules can be used for recognition of specic 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 stability 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 difculty in producing stable QD-biomolecule complexes. Developments have stressed the importance of adequate surface modications in developing luminescent QDs for labeling in bioanalysis and diagnostics, as tags for protein and DNA immunoassays or as biocompatible 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

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

O P

QD

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

Trends

4.1. Fluorescence-based transduction As the luminescence of QDs is very sensitive to the surface 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 efciency of the core electron-hole recombination [32]. This has been the basis of the increase in research activity on the development of novel optical sensors based on QD probes. Following this approach, Cd-based QDs have been reported 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 nonradiative recombination of charge carriers. A similar photoluminescence-activation effect (attributed to passivation of surface trap sites that are either being lled 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 sensing quenching strategies (based on the quenching by the analyte that affects the luminescence emission of the nanoparticle) have been proposed. Quenching mechanisms to explain how metal ions quench uorescence of QDs include inner lter effects, non-radiative recombination pathways, electron-transfer processes and ion-binding interactions. Measurement of the luminescence-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 evaluated 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 rst references to the use of luminescent QDs as selective ion probes in aqueous samples. Isarov and Chrysochoos [36] observed that the addition of Cu(II) perchlorate in 2-propanol to CdS nanoparticles 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 that

Fluorescence quenching

Fluorescence quenching

quenching quenching quenching quenching quenching-enhancement quenching quenching quenching enhancement quenching-enhancement 3.2 nM 10 nM 70 nM 0.19 ng/mL 0.1 lM 1.1 lM 5.0 nM Water Water Water Water Water Methanol Methanol Water Water Gas media CdSe CdSe-ZnS CdSe CdTe CdTe CdS CdSe CdSe CdS CdSe Fluorescence Fluorescence Fluorescence Fluorescence Fluorescence Fluorescence Fluorescence Fluorescence Fluorescence Fluorescence

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.

[34]

[35]

[37] [38] [39] [40] [41] [42] [44] [45] [46] [47]

Ref.

Detection limit

0.5 lM

0.8 mM Zn(II) 0.1 mM Cu(II)

Phosphate buffer

Matrix

Water

Table 2. QD-based fluorescent probes for chemical determination of small molecules and ions

Polyphosphate L-cysteine Thioglycerol 2-mercaptoethane sulfonic acid Bovine serum albumin Mercaptoacetic acid + bovine serum albumin 3-mercaptopropionic acid Thioglycolic acid Polyphosphate Tert-butyl-n-(2-mercaptoethyl)-carbamate 2-mercaptoethane sulfonic acid L-cysteine Incorporated in polymer lms CdS

QD material

CdSe-ZnS

CdS

Thioglycolic + organophosphorous hydrolase

QD coating

Cly-His-Leu-Leu-Cys

Cu(II) Ag(II) Cu(II) Fe(III) Zn(II) Cu(II) Cu(II) Ag(I) Cu(II) Zn(II), Mn(II), Ni(II), Co(II) I CN CN Ag+ Triethylamine Benzylamine Paraoxon
http://www.elsevier.com/locate/trac

Analyte

Water

10 nM

Fluorescence quenching

Measuring signal

[49]

211

Trends

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

the quenching could be employed for chemical sensing of Cu ions in the organic solution. Water-soluble CdSe QDs with their surface modied with 2-mercaptoethane sulfonic acid can be used for the sensitive and selective determination of copper (II) ions in aqueous solutions, based on uorescence-quenching measurements [37]. In addition, based on the photoluminescence quenching of the nanocrystals, CdSe-ZnS QDs modied with bovine serum albumin (BSA) were investigated for the determination of copper [38], CdSe QDs modied with mercaptoacetic acid and BSA were assayed for the analysis of silver [39] and CdTe nanocrystals modied 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 complexing 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 responses. They found that zinc ions enhanced the luminescence emission of the QDs. However, other metals (e.g., calcium, magnesium, manganese, nickel and cadmium) quenched luminescence. Apart from research on QD-based uorescent sensors for ion metals, work on other chemical species (e.g., iodide [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 lter effects, non-radiative recombination pathways and electron-transfer processes. The strong, reversible adsorption of negativelycharged CN onto the QD surface, with the consequent

increased location due to compression of the electronwave 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 modied with tert-butyl-n-(2-mercaptoethyl)-carbamate, has been proposed for the selective, sensitive determination of free cyanide in methanol after a photoactivation of the QDs [44]. In a further work [45], the authors reported the synthesis of water-soluble luminescent CdSe QDs, surfacemodied with 2-mercaptoethane sulfonate, for the selective determination of free cyanide in aqueous solution (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 uorescent 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 photoluminescence. 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 modied with L-cysteine. The authors showed detection limits as low as 5.0 109 M. They proposed that the uorescence-enhancement effect could be attributed to the formation of a complex between silver ions and the RS- groups adsorbed on the surface of the modied QDs, which resulted in the creation of radiative centers 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 incorporated into polymer thin lms is reversibly enhanced or quenched by the presence of certain gases in the

C N-

CdSe

C N-

CdSe
C N-

300 200
IF + CN -

300 200
IF

100 0 450

100 0 450

550 650 Wavelength, nm

550 650 Wavelength, nm

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

212

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

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

Trends

environment. After QD synthesis, photostimulation was found to be necessary to obtain a stabilized emission prole and to provide reliable responses to the presence of the gases. This effect, shown in Fig. 4, was also reported 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 surface 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 together with organophosphorous hydrolase (OPH) in a thin lm prepared by the layer-by-layer technique. The presence of paraoxon in the sample solution was detected by changes in the photoluminescence emission of the QDs, attributed to an interaction of the analyte with the OPH included in the sensing lm, changing its conformation. Following this approach, the synthesis of (CdSe)ZnS nanocrystals and their conjugation with organophosphorous hydrolase (through electrostatic interaction between negatively-charged QD surfaces and the positively-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 MichaelisMenten equation. This result indicated that the quenching was caused by the conformational change in the enzyme, which was conrmed by gas-chromatography measurements. Although such a strategy of QD-surface bioconjugation 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-specic, DNA-binding proteins that act as luminescent labels for different applications (e.g., multicolor gene mapping on the nm scale) [50]. Moreover, uorescence quenching of water-soluble CdSe QDs, surface modied with mercaptoacetic acid, has also been used to develop a uorescence 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 helix structure of the DNA in a non-intercalative way, resulting in the observed deactivation of the luminescence emission of the QDs. 4.2. Fluorescence (or Fo rster) resonance energy-transfer-based sensors Energy-transfer mechanisms have been widely used in different elds and are the basis of a new generation of

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

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

213

Trends

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

luminescent sensors [52]. In this context, the capability of tailoring (via size) QD-photoemission properties should allow efcient energy transfer with a number of conventional organic dyes, thus suggesting the use of the nanoparticles in sensor or chemical assay applications based on photochemically-induced uorescence (or Fo rster) resonance-energy-transfer (FRET) mechanisms. However, the QD emission spectrum is narrower and more symmetric than the emission from conventional organic uorophores, making it much easier to distinguish the emission of the donor from that of the acceptor. Moreover, the high quantum yields of QDs make energy transfer very efcient. Several studies have already conrmed that QDs are excellent candidates for use in the design of novel FRETbased strategies. As an example, specic binding of different 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 specic interactions between both proteins, strong enhancement of the acceptor-dye uorescence was observed [53]. In a more fundamental study, conjugation of BSA with luminescent CdTe nanoparticles (capped with Lcysteine) resulted in a signicant increase in the CdTe uorescent emission, attributed to an efcient resonance-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-FRETbased optical sensors have been published so far. Luminescent CdSe QDs have been used as energy donors in developing a competitive FRET assay for maltose [55] (see Fig. 5). Semiconductor nanoparticles bioconjugated to different maltose-binding proteins, formed using a non-covalent self-assembly scheme, act as the resonance-energy-transfer donors, while non-uorescent 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 uorescence. When maltose is present, it replaces the cyclodextrin complexes, and the QD uorescence recovers [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 modied with a thiolated oligonucleotide, 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 telomerase, during the progression of the telomerization, the 214
http://www.elsevier.com/locate/trac

(3 ita 50 tio nm n )

ex c

FRET Quenching
Dy
CD

QD
a + maltose

MBP

(3 ita 50 tio nm n )

ex c

Fluorescence

Dy
CD

QD
b

MBP

Figure 5. Schematic diagram of the quantum-dot based FRET maltose sensor (adapted from reference [56]). QDs conjugated to around 10 maltose-binding proteins function as the FRET donors. Nonuorescent dyes bound to a cyclodextrin serve as the acceptors and in the absence of maltose are lling the protein binding sites resulting in a quenching of the luminescence. When maltose is present, it removes the cyclodextrin-dye complex and the uorescence is recovered. MBP: maltose binding protein, CD: cyclodextrin.

uorescence emission from the QDs at 560 nm decreases with 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 functionalized 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 functions 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 molecule [4-(4 0 -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 uorescent signal is observed. After adding the target DNA sequence, the MB structure opens. Since QD and quencher

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

Trends

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 uorophores. The application of water-soluble ZnS nanoparticles, surface-modied with sodium thioglycolate, as uorescence probes has been described for specic 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 surface-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 results 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 solution-phase, nanoscale, sensing assemblies for detecting the explosive 2,4,6-trinitrotoluene (TNT) in aqueous environments based on FRET measurements [60]. The presence of TNT was detected by displacing the dyelabeled analogue bonded to the QD surface, resulting in the elimination of FRET and in the concentrationdependent 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 efciency is still inherently low compared to that of conventional dyes (due to the comparatively large size of QDs, it is too difcult to secure close enough proximity for FRET to occur efciently). However, several studies have been carried out in order to gain a better understanding of the process, showing that enhanced efciency 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 dyelabeled proteins to the QD surface, the overall FRET signal was improved substantially over a simple one donor-to-one acceptor FRET pair [61]. Efciency was further enhanced by increasing the number of dye acceptors in the QD bioconjugate, where QDs functioned as efcient energy donors [61]. Furthermore, the large size of QD uorophores, compared to organic dyes, allowed design of congurations where, for example, multiple acceptors could coordinate around and interact with a single QD donor. This strategy, already demonstrated for multiple detection in immunoassays [62], suggests the possibility of achieving mutianalyte optosensing 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 surfaceplasmon resonance (SPR). Redox transformations

occurring on chemically-modied surfaces may signicantly 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 nanoparticles in an Au-nanoparticle/CdS-QD array (coating an Au/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 Ausemiconductor-nanoparticle array and so activate photoelectrochemical functions suggests that using SPR spectroscopy combined with surface-modied QDs could provide an alternative tool for new SPR (bio)sensor probes. 4.4. Phosphorescence transduction Only uorescence transduction has so far been employed for photoluminescence sensing in combination with QDs. However, investigation of the luminescence properties of QDs is slowly expanding into phosphorescence, a detection 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-dened 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 roomtemperature phosphorescence (RTP) emission, although the origin of the luminescence and the emission mechanism is not yet understood [66]. Moore et al. from Mercer University have also reported phosphorescence emission from aqueous mixed sulde QD matrices, QD-CdxZn1-xS, doped with manganese(II) [67]. The authors evaluated the impact of matrix composition on the QD phosphorescence ($590 nm). They found that the observed RTP intensity for the CdxZn1xS:Mn QDs was very sensitive to matrix composition (e.g., the 590 nm emission increased with the Zn concentration 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 towards developing useful optosensing approaches [68] consists of immobilizing those QDs in appropriate solid supports to fabricate active solid phases for working in owing solutions [65]. In this context, sol-gel materials have been demonstrated to be especially suited to the development of luminescent optical sensors by trapping the indicator
http://www.elsevier.com/locate/trac

215

Trends

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

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 luminescent nanocrystals. 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 approaches 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 concentrators or as active media in tunable lasers [73]). However, these sol-gel materials, doped with luminescent QDs, are also expected to be also for optosensing applications in the near future. A related approach is to incorporate QDs into molecularly imprinted polymers (MIPs) [74], which act as articial 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-linkers and 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 uorescence-energy-transfer processes between the QDs and the template molecules. The approach has been successfully tested for caffeine detection, although additional 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 attempted 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. 216
http://www.elsevier.com/locate/trac

ii Quantum dots have been demonstrated to be especially 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 interaction with QD particles. Of course, those methods are not 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 surface, 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 uorescence 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 has not yet been demonstrated.

5. Conclusions and future prospects The popularity of QDs as photoluminescent probes for optical sensing is steadily increasing, as researchers move to exploit the unique properties of this new class of luminophores. Optosensing technologies will probably combine the important advantages of QDs with ow-analysis techniques and perhaps bre-optic instrumentation. Chemical-sensing developments will benet 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 expected. Chemical-surface modications of the QDs have still to be perfected in order to enhance the selectivity of the systems and to prot from their favorable emission features. In this context, the conjugation of selective reagents to the surface of luminescent 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 uorescent probes for sensing applications. Moreover, approaches such as the combination of the nanoparticles with energy-transfer processes, phosphorescence 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 ow-through optical sensing or ber-optic-based sensing applications.

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

Trends
[32] D.E. Moore, K. Patel, Langmuir 17 (2001) 2541. [33] K. Sooklal, B.S. Cullum, S.M. Angel, C.J. Murphy, J. Phys. Chem. 100 (1996) 4551. [34] K.M. Gatas-Asfura, R.M. Leblanc, Chem. Commun. (2003) 2684. [35] Y. Chen, Z. Rosenzweig, Anal. Chem. 74 (2002) 5132. [36] A.V. Isarov, J. Chrysochoos, Langmuir 13 (1997) 3142. [37] M.T. Fernandez-Arguelles, W.J. Jin, J.M. Costa-Fernandez, R. Pereiro, A. Sanz-Medel, Anal. Chim. Acta (2005) 20. [38] H.Y. Xie, J.G. Liang, Z.L. Zhang, Y. Liu, Z.K. He, D.W. Pan, Spectrochim. Acta Part A 60 (2004) 2527. [39] J.G. Liang, X.P. Ai, Z.K. He, D.W. Pang, Analyst (Cambridge, UK) 129 (2004) 610. [40] C. Bo, Z. Ping, Anal. Bioanal. Chem. 381 (2005) 986. [41] J. Li, D. Bao, X. Hong, D. Li, J. Li, Y. Bai, T. Li, Colloids Surf A 257 258 (2005) 267. [42] J.R. Lakowicz, I. Gryczynski, Z. Gryczynski, C.J. Murphy, J. Phys. Chem. B 103 (1999) 7613. [43] S.K. Sarkar, N. Chandrasekharan, S. Gorer, G. Hodes, Appl. Phys. Lett. 81 (2002) 5045. [44] W.J. Jin, J.M. Costa-Fernandez, R. Pereiro, A. Sanz-Medel, Anal. Chim. Acta 522 (2004) 1. ndez-Arguelles, J.M. Costa-Fernandez, [45] W.J. Jin, M.T. Ferna R. Pereiro, A. Sanz-Medel, Chem. Commun. (2005) 883. [46] J.-L. Chen, C.-Q. Zhu, Anal. Chim. Acta 546 (2005) 147. [47] A.Y. Nazzal, L. Qu, X. Peng, M. Xiao, Nano Lett. 3 (2003) 819. s-Asfura, S.V. Mello, G. Crespo, [48] C.A. Constantine, K.M. Gatta V. Rastogi, T.C. Cheng, J.J. DeFrank, R.M. Leblanc, J. Phys. Chem. B 107 (2003) 13762. [49] X. Ji, J. Zheng, J. Xu, V.K. Rastogi, T.-C. Cheng, J.J. DeFrank, R.M. Leblanc, J. Phys. Chem. B 109 (2005) 3793. [50] D. Gerion, W.J. Parak, S.C. Williams, D. Zanchet, C.M. Michael, A.P. Alivisatos, J. Am. Chem. Soc. 124 (2002) 7070. [51] L.-Y. Wang, L. Wang, F. Gao, Z.-Y. Yu, Z.-M. Wu, Analyst (Cambridge, UK) 127 (2002) 977. [52] J.M. Traviesa, J.M. Costa, R. Pereiro, A. Sanz-Medel, Talanta 62 (2004) 827. [53] D.M. Willard, L.L. Carillo, J. Jung, A. Van Orden, Nano Lett. 1 (2001) 469. [54] N.N. Mamedova, N.A. Kotov, A.L. Rogach, J. Studer, Nano Lett. 1 (2001) 281. [55] H. Mattoussi, J.M. Mauro, E.R. Goldman, G.P. Anderson, V.C. Sundar, F.V. Mikulec, M.G. Bawendi, J. Am. Chem. Soc. 122 (2000) 12142. [56] I.L. Medintz, A.R. Clapp, H. Mattoussi, E.R. Goldman, B. Fisher, J.M. Mauro, Nat. Mater. 2 (2003) 630. [57] F. Patolski, R. Gill, Y. Weizmann, T. Mokari, U. Banin, I. Willner, J. Am. Chem. Soc. 125 (2003) 13918. [58] J.H. Kim, D. Morikis, M. Ozkan, Sens. Actuators B 102 (2004) 315. [59] L.-Y. Wang, X.-W. Kan, M.-C. Zhang, C.-Q. Zhu, L. Wang, Analyst (Cambridge, UK) 127 (2002) 1531. [60] E.R. Goldman, I.L. Medintz, J.L. Whitley, A. Hayhurst, A.R. Clapp, H.T. Uyeda, J.R. Deschamps, M.E. Lassman, H. Mattoussi, J. Am. Chem. Soc. 127 (2005) 6744. [61] A.R. Clapp, I.L. Medintz, J.M. Mauro, B.R. Fisher, M.G. Bawendi, H. Mattoussi, J. Am. Chem. Soc. 126 (2004) 301. [62] E.R. Goldman, A.R. Clapp, G.P. Anderson, H.T. Uyeda, J.M. Mauro, I.L. Medintz, H. Mattoussi, Anal. Chem. 76 (2004) 684. [63] M. Zayats, A.B. Kharitonov, S.P. Pogorelova, O. Lioubashevski E. Katz, I. Willner, J. Am. Chem. Soc. 125 (2003) 16006. [64] J. Kuijt, F. Ariese, U.A.Th. Brinkman, C. Gooijer, Anal. Chim. Acta 488 (2003) 135. [65] A. Sanz-Medel, Anal. Chim. Acta 283 (1993) 367. [66] P. Yang, M.K. Lu, C.F. Song, G.J. Zhou, D. Xu, D.R. Yan, Inorg. Chem. Commun. 5 (2002) 187. [67] http://chemistry.mercer.edu/dem/demres.html . [68] J. Ruzicka, E.M. Hansen, Anal. Chim. Acta 173 (1985) 3.

In brief, the future of QDs for optical sensing looks bright, as their analytical potential in the eld now starts to be realized.

Acknowledgement Financial support from the EU Project SWIFT-WFD (Contract SSPI-CT-2003-502492) and MAT200309074-C02 (Feder Programme and Ministerio de Ciencia a, Spain) is gratefully acknowledged. y Tecnolog References
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] C.M. Niemeyer, Angew. Chem. Int. Ed. Engl. 40 (2001) 4128. A.P. Alivisatos, Science (Washington, DC) 271 (1996) 933. C.J. Murphy, J.L. Coffer, Appl. Spectrosc. 56 (2002) 16A. H. Weller, Angew. Chem. Int. Ed. Engl. 32 (1993) 41. L.E. Brus, J. Chem. Phys. 79 (1983) 5566. L.E. Brus, J. Chem. Phys. 80 (1984) 4403. L. Spanhel, M. Haase, H. Weller, A. Henglein, J. Am. Chem. Soc. 109 (1987) 5649. C.B. Murray, D.J. Norris, M.G. Bawendi, J. Am. Chem. Soc. 115 (1993) 8706. S. Chaudhary, M. Ozkan, W.C.W. Chan, Appl. Phys. Lett. 84 (2004) 2925. V. Colin, M.C. Schlamp, A.P. Alivisatos, Nature (London) 370 (1994) 374. M. Bruchez, M. Moronne, P. Gin, S. Weiss, A.P. Alivisatos, Science (Washington, DC) 281 (1998) 2013. W.C.W. Chan, S.M. Nie, Science (Washington, DC) 281 (1998) 2016. Z.A. Peng, X.G. Peng, J. Am. Chem. Soc. 123 (2001) 183. X. Peng, M.C. Schlamp, A.V. Kadavanich, A.P. Alivisatos, J. Am. Chem. Soc. 119 (1997) 7019. A.R. Kortan, R. Hull, R.L. Opila, M.G. Bawendi, M.L. Steigerwald, P.J. Carroll, L.E. Brus, J. Am. Chem. Soc. 112 (1990) 1327. C.J. Murphy, Anal. Chem. 74 (2002) 520A. L.E. Brus, J. Chem. Phys. 90 (1986) 2555. C.B. Murray, D.J. Norris, M.G. Bawendi, J. Am. Chem. Soc. 115 (1993) 8706. J.E. Bowen-Katari, V.L. Colvin, A.P. Alivisatos, J. Phys. Chem. 98 (1994) 411. P. Reiss, G. Quemard, S. Carayon, J. Bleuse, F. Chandezon, A. Pron, Mater. Chem. Phys. 84 (2004) 10. B.O. Dabbousi, J. Rodriguez-Viejo, F.V. Mikulec, J.R. Heine, H. Mattoussi, R. Ober, K.F. Jensen, M.G. Bawendi, J. Phys. Chem. B 101 (1997) 9463. L. Qu, X. Peng, J. Am. Chem. Soc. 124 (2002) 2049. M. Henini, S. Sanguinetti, L. Brusaferri, E. Grilli, M. Guzzi, M.D. Upward, P. Moriarty, P.H. Beton, Microelectron. J. 28 (1997) 933. A.L. Rogach, A. Kornowski, M. Gao, A. Eychmu ller, H. Weller, J. Phys. Chem. B 103 (1999) 3065. B. Ludolph, M.A. Malik, P. OBrien, N. Revaprasadu, Chem. Commun. (1998) 1849. D.J. Crouch, P. OBrien, M.A. Malik, P.J. Skabara, S.P. Wright, Chem. Commun. (2003) 1454. A.J. Sutherland, Curr. Opin. Solid State Mater. Sci. 6 (2002) 365. W.C.W. Chan, D.J. Maxwell, X. Gao, R.E. Bailey, M. Han, S. Nie, Curr. Opin. Biotechnol. 13 (2002) 40. A.M. Smith, S. Nie, Analyst (Cambridge, UK) 129 (2004) 672. J. Riegler, T. Nann, Anal. Bioanal. Chem. 379 (2004) 913. P. Alivisatos, Nat. Biotechnol. 22 (2004) 47.

[22] [23]

[24] [25] [26] [27] [28] [29] [30] [31]

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

217

Trends
ndez, A. Sanz Medel, Anal. Chim. Acta 407 [69] J.M. Costa Ferna (2000) 61. [70] S.T. Selvan, C. Bullen, M. Ashokkumar, P. Mulvaney, Adv. Mater. 13 (2001) 985. [71] C. Bullen, P. Mulvaney, C. Sada, M. Ferrari, A. Chiasera, A. Martucci, J. Mater. Chem. 14 (2004) 1112.

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


[72] H. Jiang, X.Y.J. Che, M. Wang, F. Kong, Ceramics Int. 30 (2004) 1685. [73] R. Reisfeld, Opt. Mater. (Amsterdam) 16 (2001) 1. [74] K. Yano, I. Karube, Trends Anal. Chem. 18 (1999) 199. [75] C.I. Lin, A.K. Joseph, C.K. Chang, Y.D. Lee, Biosens. Bioelectron. 20 (2004) 127.

218

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