Introduction

The past 20 years have seen growing research activity in optical sensing. In particular, the measurement of the photoluminescence from a luminophor sensitive to a given analyte is a well-established approach commonly used for fabrication of reliable optical sensors, mainly because of the high sensitivity and selectivity typical of such systems. The current demand for new optosensors for practical applications has led to a search for novel robust luminescent probes. In this vein, the wealth of novel physical, chemical, and biological behaviour that occurs on the nanometre scale has resulted in increasing interest in the synthesis and application of nanomaterials as an improved alternative to conventional organic luminescent indicators. Among these nanostructures, luminescent semiconductor nanocrystals, or quantum dots, “QD”, have many fascinating optoelectronic properties [1] with substantial promise for the construction of a new generation of optical probes.

Quantum dots are colloidal nanocrystalline semiconductors, roughly spherical, with particle diameters typically ranging from 1 to 12 nm [1, 2]. At such small sizes (close to or smaller than the dimensions of the exciton Bohr radius within the corresponding bulk material) these nanostructured materials behave differently from bulk solids, because of quantum-confinement effects [2]. In fact, when synthesized at the nanometer size, and after adequate surface protection (e.g. by capping the surface of the QD with appropriate protective ligands), these compounds develop intense and long-lasting luminescent emission with very narrow emission bandwidths (full width at half-maximum of approximately 15–40 nm). Quantum dots typically have higher fluorescence quantum yields and better chemical and photoluminescence stability than conventional organic fluorophores. Furthermore, these nanocrystals have size-dependent tunable photoluminescence emission. In fact, QD can be made to emit luminescence from the ultraviolet to the near-infrared spectral region. The frequency of the light emitted by a specific quantum dot is related directly to its size; smaller particles tend to emit higher-energy (shorter wavelength) radiation (Fig. 1).

Fig. 1
figure 1

Fluorescence emission of colloidal CdSe nanocrystals (after excitation at 400 nm) with an indication of particle size. Size-tuneable fluorescence spectra of the different sized CdSe QD are shown below

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In 1998 Alivisatos’s and Nie’s groups simultaneously demonstrated that water-soluble and biocompatible quantum dots could be prepared by appropriate surface modification of the nanocrystals [3, 4]. They also showed the great potential of such nanocrystals to be used as highly sensitive fluorescent biomarkers. Since those pioneering developments, crystal growth and post-growth processing technology have developed to the extent that it is now easily possible to fabricate high-quality luminescent quantum dots [5] and to modify their surface by conjugation with appropriate functional molecules [6], thus enabling their use in a wide variety of (bio)chemical applications.

During the last few years, much work has been directed toward the synthesis and use of photoluminescent quantum dots for biochemical applications (as labels in bioanalysis and diagnostics, as tags for protein and DNA immunoassays, or as biocompatible labels for in-vivo imaging studies) [7–10]. Quantum dots also have unique attributes that make them superior to commercially available organic dyes when used for optical sensing [11]. Analytical chemists have, therefore, also started to explore these nanomaterials for development of a new generation of luminescence optical probes. This brief article summarizes some of the very recent developments and trends reported so far with the objective of expanding the field of application of QD for optical sensing purposes.

QD surface interaction-based optical sensors

Fundamental studies dealing with the characterization of the optoelectronic properties of quantum dots have revealed that luminescence of QD is very sensitive to their surface states. Thus, it is reasonable to expect that eventual chemical or physical interactions between a given chemical species with the surface of the nanoparticles would result in changes of the QD surface charges, and would affect the QD photoluminescence emission very significantly [12]. Measurement of the fluorescence changes that occur after interaction of the target analyte with the nanoparticle surface has, in fact, been the basis of many of the QD-based optical probes reported so far.

Following this approach, luminescence enhancement of water-soluble CdS quantum dots surface-modified with L-cysteine was proposed very recently for optical sensing of trace levels of silver ion [13]. In this report, the authors proposed that complex formation between silver ions and the RS groups adsorbed on the surface of the modified QD gave rise to new radiative centres in the CdS/Ag-SR complex, resulting in the observed enhancement of the fluorescence.

Besides the activation effect, other proposed QD-based optical detection strategies make use of the quenching effect of an analyte on the luminescence emission of a nanoparticle. Measurement of the luminescence deactivation ratio of surface-modified water-soluble quantum dots has recently been proposed for optical monitoring of several cations, for example Zn(II) and Cu(II) [12, 14], of some toxic anions, for example cyanide [15], or for reversible optosensing of some reactive gas molecules (e.g. triethylamine and benzylamine) [16]. Several quenching mechanisms (including inner filter effects, nonradiative recombination pathways, electron-transfer processes and ion-binding interactions) have been proposed to explain how the different analytes quench the fluorescence from the QD.

The reported methods based on the detection of the luminescence changes that occur after interaction of the analyte with the QD surface are very simple and easy to develop, and some have very high sensitivity. Unfortunately, these methods seem to be restricted to sensing only a few reactive small molecules or ions (those able to interact with the QD surface). Moreover, lack of selectivity, low stability of the QD in aqueous media, and limited applicability to real sample analysis (because of the special experimental conditions required) are among the limitations of some of the optosensors proposed so far.

The conjugation of selective reagents to the surface of luminescent QD (a strategy well established for imaging, immunoassay, and labelling applications in biological science) has also been used for development of QD-based fluorescent probes. As an example, water-soluble CdSe QD surface-functionalized with thioglycolic acid have been synthesized and incorporated together with organophosphorus hydrolase (OPH) in a thin film. Exposure of this sensing film to the pesticide paraoxon resulted in detectable changes in the photoluminescence emission of the QD, attributed to selective interaction of the analyte with the immobilized OPH [17]. Although this strategy of QD surface bioconjugation has not yet been frequently exploited for sensing purposes, it clearly it has great potential for further development.

Fluorescence resonance energy-transfer-based sensors

The potential uses of QD in optical sensing applications could expand because of their ability to function as energy-transfer donors in fluorescence resonance energy-transfer (FRET) mechanisms. The ability to tailor (via size) the QD photoemission properties would enable efficient energy transfer with a wide number of conventional organic dyes. The high quantum yields of QD make energy transfer very efficient. Furthermore, the QD emission spectrum is narrower and more symmetric than the emission from conventional organic fluorophores, making it much easier to distinguish the emission of the donor from that of the acceptor. Several studies have already confirmed that QD are excellent donors in FRET-based assays. As an example, specific binding of different proteins was observed via FRET between a CdSe–ZnS QD donor, attached to one of the proteins, and organic acceptor dyes attached to the other protein under study; this resulted in strong enhancement of the dye fluorescence that was well resolved from the QD absorption spectrum [18].

Insight gained from fundamental studies of FRET in QD–protein–dye bioconjugates has been used to build a prototype QD nanoscale sensing strategy. Luminescent CdSe QD have been used as energy donors in the development of a prototype QD-based sensor for maltose sensing in solution based on competitive FRET assay [19]. The assays monitored the competition between cyclodextrin (conjugated to an acceptor dye) and maltose in binding to receptor proteins attached to the QD. Semiconductor quantum dots were bioconjugated to different maltose-binding proteins (MBP). The QD–MBP conjugates acted as the resonance energy-transfer donors and nonfluorescent dyes bound to cyclodextrin served as the energy-transfer acceptors. In the absence of maltose, cyclodextrin–dye complexes occupy the protein-binding sites. Energy transfer from the QD to the dyes quenches the QD fluorescence. When maltose is present it replaces the cyclodextrin complexes and the QD fluorescence is recovered (Fig. 2).

Fig. 2
figure 2

Prototype quantum-dot-based FRET maltose sensor (adapted from Ref. [19]). A 555-nm emitting QD, conjugated to approximately 15–20 maltose-binding proteins (MBP), function as the FRET donor. Non-fluorescent dyes QSY9 (absorption max. at approx. 565 nm) bound to a cyclodextrin (CD) fill the protein binding sites and serve as the acceptors quenching the QD-MBP luminescence emission. When maltose is present it displaces the cyclodextrin–dye complex and the fluorescence is recovered

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It should be mentioned that FRET efficiency obtained using QD as donor species is still inherently low compared with use of conventional dyes. This is because the large size of QD makes it almost impossible to bring the acceptor into close-enough proximity to the donor for FRET to occur efficiently. Several studies have, however, been conducted to enable better understanding of the process. It has been demonstrated that enhanced FRET efficiency can be obtained by a careful design of the QD bioconjugation scheme.

Outlook

Optical sensing developments will benefit from the continuous advances in the science of quantum dots. Despite recent progress, however, much work must still be done to achieve reproducible and robust surface functionalization and to develop flexible bioconjugation techniques, searching for enhancement of the stability and selectivity of the QD-based optosensors.

Although only fluorescence transduction has been employed in combination with QD for optical sensing, investigation of the luminescence properties of QD has slowly expanded to phosphorescence detection also [20] (providing several well-known advantages for the design of reliable and robust optical sensors). Although very preliminary, these studies open a new route to novel and powerful transduction schemes that will contribute to expanding the application field of QD for optical sensing.

Most of the work performed so far have been restricted to solution-sensing assays. Quantum dots should, however, be integrated into appropriate solid supports, a process that has only just begun, to develop reliable optical sensors for true direct and “in situ” monitoring. In this vein, several approaches have been already proposed, for example synthesis of sol–gel materials doped with QD [21] or incorporation of the luminescent quantum dots into molecularly imprinted polymers (MIP) [22] which act as artificial receptors/antibodies with tailor-made selectivity for the template molecule. In a very recent approach, different MIP incorporating CdSe/ZnS core-shell QD derivatized with 4-vinylpyridine were synthesized and successfully evaluated for caffeine detection [22]. These processes are not simple, because changes in solvent polarity or surface reactions during the polymerization would result in an undesirable quenching of the QD luminescence.

The unique optical properties of quantum dots also make them ideal labels for use in simultaneous monitoring of several analytes. This has already been demonstrated for multiple detection of several toxins in immunoassays using four different antibodies conjugated to four QD of different sizes [23]. By following this approach, appropriate surface-modified QD of different sizes (which can be excited with a single excitation source but which have different well-resolved emission wavelength maxima) and selective to different analytes could be used in reliable multiple analyte optosensing strategies.

Clearly, the concept of QD as luminescent probes for optical sensing developments has become a reality. The potential of QD as optical probes has just begun to be realized and the analytical community will continue exploiting these nanomaterials, which have a very promising future in optical sensing.