Anal Bioanal Chem (2010) 396:229–240 DOI 10.1007/s00216-009-3033-0

REVIEW

Bioconjugated quantum dots as fluorescent probes for bioanalytical applications Manuela F. Frasco & Nikos Chaniotakis

Received: 30 June 2009 / Revised: 24 July 2009 / Accepted: 30 July 2009 / Published online: 28 August 2009 # Springer-Verlag 2009

Abstract Quantum dots (QDs) are inorganic semiconductor nanocrystals that have unique optoelectronic properties responsible for bringing together multidisciplinary research to impel their potential bioanalytical applications. In recent years, the many remarkable optical properties of QDs have been combined with the ability to make them increasingly biocompatible and specific to the target. With this great development, QDs hold particular promise as the next generation of fluorescent probes. This review describes the developments in functionalizing QDs making use of different bioconjugation and capping approaches. The progress offered by QDs is evidenced by examples on QD-based biosensing, biolabeling, and delivery of therapeutic agents. In the near future, QD technology still faces some challenges towards the envisioned broad bioanalytical purposes. Keywords Quantum dots . Bioconjugation . Fluorescence . Biosensing . Biolabeling and imaging

Introduction The unique photophysical properties of colloidal luminescent semiconductor nanocrystals, or quantum dots (QDs), attributed to quantum confinement effects have elicited intensive research for sensing, labeling, and imaging applications. QDs have high quantum yields, broad absorpM. F. Frasco : N. Chaniotakis (*) Laboratory of Analytical Chemistry, Department of Chemistry, University of Crete, Vassilika Voutes, 71003 Iraklion, Crete, Greece e-mail: [email protected]

tion spectra, narrow size-tunable emissions, and are resistant to photobleaching as well as to chemical degradation [1]. The smaller the QDs, the more blueshifted their fluorescence, and by synthesizing QDs of different sizes, one can obtain a range of colors. The synthesis and engineering of QDs with different semiconductor materials have expanded the range of possible emission wavelengths from the visible to the red and infrared regions (e.g., CdSe may be size-tuned to emit in the 450–650-nm range, CdTe can emit in the 500–750-nm range, whereas InAs or PbSe can emit above 800 nm) [2]. The simultaneous detection of multiple targets at different wavelengths with a single excitation wavelength is thus a possibility distinctively linked to the optical properties of QDs (Fig. 1). Owing to the well-known critical influence of the surface of QDs on the photoluminescence, a common procedure involves the overgrowth of an additional passivating inorganic shell of a semiconductor material with a larger bandgap (e.g., CdSe/ZnS or InAs/ZnSe core–shell nanocomposites). In core–shell structured QDs, the overcoating improves the photoluminescence quantum yields by passivating surface nonradiative recombination sites. Moreover, particles passivated with inorganic shell structures are more robust and have greater tolerance to processing conditions, which is very appealing for different fields of application of QDs [3]. The exceptional features of QDs are advantageous for sensitive and specific fluorescence detections in countless nanoassemblies using QDs as probes, as opposed to organic and protein-based fluorophores. Many of the QD-based optical probes reported so far rely on changes in the fluorescence occurring when the target analyte interacts with the QD surface. Interactions occurring at the surface of the QDs change the efficiency of the radiative recombination, leading to either photoluminescence activation or quenching. In general, a fluorescence

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Emission

Absorbance

a

Wavelength Near-infrared Size

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Fig. 1 a Size-tunable emission spectra of quantum dots (QDs); the dashed line exemplifies the absorption spectrum of the QD emitting at the lower wavelength. b Size-tunable fluorescence properties of QDs: ten distinguishable emission colors of CdSe/ZnS QDs excited with a near-UV lamp. From left to right (blue to red), the emission maxima are located at 443, 473, 481, 500, 518, 543, 565, 587, 610, and 655 nm. (Reprinted with permission from Macmillan Publishers Ltd (Nature Biotechnology) [28], copyright 2001)

quenching can be attributed to inner-filter effects, nonradiative recombination pathways, and electron transfer processes, whereas fluorescence enhancement can be ascribed to passivation of trap states or defects on the surface of the QDs [4]. As it will be discussed in the following sections, the sensitivity and selectivity towards the target can be achieved by covering QDs with particular ligands. Thus, knowledge of the surface chemistry of QDs together with the ability to functionalize them elicited the development of simple and easy methods that have been applied for sensing small molecules or ions [5–7]. QDs have been successfully adopted in fluorescence (Förster) resonance energy transfer (FRET)-based studies. FRET is a powerful technique for probing very small changes in the distance between donor and acceptor fluorophores because the efficiency of energy flow depends on the distance, spectral overlap, and relative orientation of both the donor and the acceptor [8]. The broad excitation spectra and large absorption cross section of QDs, combined with the narrower excitation spectra of acceptor

dyes, enable one to choose the most appropriate excitation wavelength to reduce unwanted direct excitation of the acceptor and thus increase FRET efficiency. Moreover, since the emission spectrum of the QD donor is narrow, symmetric, and has no red tail, the cross talk with the fluorescence spectrum of the acceptor is minimized. FRET efficiency can also be enhanced because multiple acceptors can interact at the surface of a single QD, and the spectral overlap can be adjusted by changing the size of the QDs [9]. Combined with these favorable properties of QDs, ideal platforms have been established for the sensitive detection of molecular events occurring upon interaction with a particular target [10, 11] (Fig. 2a). QDs can emit light in the near-infrared region of the spectrum, allowing a lower background signal from biological samples and a deeper tissue penetration (e.g., near-infrared-emitting CdTe/CdSe QDs used for in vivo fluorescence imaging of lymph nodes at up to 1-cm depth) [12]. However, one of the most propagated disadvantages of QDs for in vivo imaging refers to the necessary blue light for efficient excitation. Since blue light does not have good tissue penetration and excites endogenous fluorophores, the outcome for in vivo imaging is low excitation efficiency and high background [13]. Recently, to tackle this problem, QDs were successfully used as energy acceptors in bioluminescence resonance energy transfer (BRET), a process that occurs without an excitation source, in contrast to FRET (Fig. 2b). QDs covalently conjugated with Renilla reniformis luciferase (Luc8) become a bioluminescence probe because the luminescent light produced during the chemical oxidation of coelenterazine in the presence of oxygen by Luc8 is efficiently transferred to the QDs [14]. QDs can also be used in a process similar to BRET designated chemiluminescence resonance energy transfer (CRET), which involves the nonradiative transfer of energy from a chemiluminescent donor to QDs as energy acceptors. QD-CRET donor conjugates have been investigated using a system based on the oxidation of the luminescent substrate luminol (in a luminol–hydrogen peroxide system) catalyzed by horseradish peroxidase (HRP) [15]. QDs were covalently coupled to the catalyst HRP, allowing an efficient CRET between luminol and the QDs. Moreover, to potentiate the use of this system in immunoassays, QDs were first electrostatically linked to bovine serum albumin (BSA) and HRP covalently conjugated with BSA antibody (anti-BSA) so that CRET occurred when anti-BSA–HRP specifically immunoreacted with BSA–QD (Fig. 2c). In the following sections, we start by presenting the developments in functionalizing QDs, urged on by the challenges currently faced in bioanalysis. The progressive knowledge on the surface chemistry aims at improving biocompatibility, sensitivity, and specificity features, pro-

Bioconjugated quantum dots as fluorescent probes for bioanalytical applications

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Fig. 2 QD-based fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), and chemiluminescence resonance energy transfer (CRET) nanosensors. a QD donor conjugated with dye-acceptor-labeled proteins, with an efficient FRET ensured by site-specific protein labeling and controlled orientation of the protein on the QD providing homogeneous donor–acceptor separation distances. b QDs conjugated with a BRET donor and transfer of bioluminescence energy to the QDs, e.g., bioluminescence energy of Renilla reniformis luciferase catalyzed oxidation of coelenterazine [14]. c Immunoassay-based transfer of chemiluminescence energy from the CRET donor to the QD acceptor, e.g.,

horseradish peroxidase (HRP)-catalyzed oxidation of luminol allows for CRET upon the immunorecognition of QD–bovine serum albumin (BSA)/BSA antibody–HRP [15]. Different types of energy transfer in QD-based biosystems are highlighted. The conjugation process is normally optimized to meet the requirements of each unique proteinQD conjugate application. In some cases a low ratio of protein to QD potentiates the fluorescent signal (e.g., in many antibody–QD conjugates), whereas other applications may require a high density of protein on the QD surface (e.g., up to ten proteins may be coupled per QD)

viding the grounds for the multitude of applications described afterwards. Subsequently, some examples illustrating the versatility of QDs as bioanalytical probes for a great variety of targets and the advantages of incorporating QDs in a number of potential nanosensors are presented. Using QDs for in vivo fluorescence applications is promising but at the same time very challenging. The multiple capabilities are once more highlighted by the literature reports on the role of QDs in providing new insights into crucial biological processes, concomitant with the labeling and imaging of clinically and pharmacologically relevant biomarkers. QD-based biosensing of diseaserelated dynamic biochemical processes and intracellular delivery of therapeutic agents are also exemplified. We conclude by giving a short overview of some limiting issues on the use of QDs for the bioanalytical purposes described herein.

types of interactions can be used to attach biological molecules to the surface of QDs, such as simple adsorption [17], through dative thiol bonding [18], or via metal-affinity coordination [19]. These functional groups displayed on the surface of QDs may participate in the subsequent attachment of other biomolecules, with the lability of the primary conjugation maintaining the main role on the stability of the final QD bioconjugate. Electrostatic self-assembly onto dihydrolipoic acid (DHLA)-capped QDs, namely, making use of a histidine tag or other highly positive charged motifs in proteins, is a well-known versatile method developed by Mattoussi’s group, pioneers in conjugating protein molecules to luminescent QDs [20, 21]. Other common approaches include the formation of covalent bonds and affinity binding chemistry [22, 23]. For the majority of sensing applications, ligand-modification concurs with the necessary yielding of water-soluble QDs, either by ligand exchange in which the stabilizing molecules are partially replaced by the sensing ligands or by ligand binding to the stabilizing shell (Fig. 3a). For biomedical labeling and imaging, the use of QDs as a substitute for organic fluorophores may represent a great breakthrough if the challenge of producing highquality water-soluble QDs active in bioconjugate reactions and with negligible toxicity is surpassed. Progress in designing various types of biocompatible coating has greatly contributed to coping with these issues. One of

Bioconjugation and capping strategies The research work presented by Chan and Nie [16] was pioneering in proposing semiconductor QDs covalently coupled to biomolecules as sensitive tools for biological detection. Since then, the surface of QDs has been engineered with ligands featuring diverse affinity and specificity towards a multitude of target analytes. Several

232 Fig. 3 Common surface modification strategies to obtain water-soluble bioconjugated QDs with functional ligands (a) or surface-capping for increased stability, biocompatibility and for multiplex sensing (b)

M.F. Frasco, N. Chaniotakis

a Direct linkage / ligand exchange Dative thiol bonding - COOH - OH - NH 2 - Oligonucleotides - Peptides - Antibodies -- -

S M

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Covalent binding COOH

NH 2

(amide bond; carbodiimide chemistry)

NH 2

SH

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b Coating / encapsulation Silanization O Si Si S O O S Si O Si O O S Si O Si

the most explored approaches is the use of silanized QDs, since a polymerized silica coating increases the stability in buffers under physiological conditions and diminishes the toxicity of the inorganic nanocrystals [24] (Fig. 3b). The embedded QDs retain their optical properties and the silica coating allows the introduction of biological functional groups. Thus, this approach is by no means incompatible with the necessary surface conjugation with relevant molecules for biological applications, as demonstrated by the successful covalent binding of oligonucleotides or for bioconjugation with immunoglobulin G (IgG)-type proteins [25–27]. Other strategies involve the encapsulation of QDs in polymer matrices. In a breakthrough report, different-sized CdSe/ZnS QDs were embedded in polystyrene beads at precisely controlled ratios, and the oligonucleotides conjugated on the surface of the polymer served as target signals [28]. With this encoded-bead approach, a multiplex DNA hybridization system was designed where the unique QD coding (based on multiple wavelengths and intensities) and target signals could be simultaneously read at the single-

Hydrophobic interaction (e.g., amphiphilic polymers)

Nanobeads

COOH NHCO O=P

CONH NHCO COOH n

bead level. CdSe/ZnS-QD-encoded polystyrene beads with immobilized human IgG allowed the effective detection of the antibody signal in solution [29]. Different capping materials have been proposed as efficient embedding agents of QDs, rendering them water-soluble, chemically stable, and biocompatible in physiological media. QDs encapsulated into amphiphilic polymers (e.g., octylamine-modified polyacrylic acid) [30, 31], chitosan [32], hyperbranched copolymer poly(ethylene glycol)-grafted polyethyleneimine [33], micelle-type coating with a gemini surfactant [34], or calcium carbonate [35] have prospective uses in various types of bioassays (Fig. 3b). There is a study based on the FRET mechanism describing the coating of QDs with amphiphilic polymer incorporating directly the acceptor dye [36]. Possessing the advantages of polymer-coated QDs, such as colloidal stability and postmodification with functional groups, this strategy increased the FRET efficiency by maintaining the acceptor and the donor at a short, fixed distance. In this way, the spectral changes of the acceptor were monitored upon analyte binding, in contrast to most FRET-based nanosensors, where detection relies on

Bioconjugated quantum dots as fluorescent probes for bioanalytical applications

specific changes of the donor-acceptor distance influenced by analyte binding.

Bioanalytical probes Nucleic acid detection Numerous works have shown that biocompatible surfacemodified QDs are viable substrates for fluorescence-based DNA detection. The specificity of the hybridization target probe is the basis of many DNA sensing approaches using different linkage strategies of the probe oligonucleotides on the surface of QDs (e.g., amide, streptavidin). The quenching/recovery of fluorescence when QDs and fluorophores, quenchers, or gold nanoparticles are brought into close proximity or spaced apart illustrates the variety of changes in the fluorescent signals used to discriminate the presence of the target DNA. One such example made use of two probes, a reporter probe labeled with an organic fluorophore and a capture probe labeled with biotin. In the presence of the target DNA, these two target-specific oligonucleotide probes originated a sandwich hybrid that could be captured by streptavidin-capped QDs [37] (Fig. 4).

Fig. 4 Single QD-based DNA nanosensors: a formation of the hybrid nanoassembly in the presence of DNA targets; b fluorescence from the organic fluorophore (Cy5) after excitation of QDs due to FRET between the QD donor and the acceptor dye; c detection of single-QD FRET signals in the presence of targets by the colocalization of QDs

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The formation of the nanoassembly enabled FRET to occur from the QD to the fluorophores in close proximity, and the system revealed some major advantages. Several sandwiched hybrids were captured by a single QD, so QDs function as nanoscaffolds, resulting in amplified signals. In addition, the presence of targets was detected by the colocalization of two fluorescent signals, and because unhybridized probes do not participate in FRET, background fluorescence was negligible. A similar principle of QD-based single-molecule coincident detection has been proposed but using two QDs with different wavelength emissions instead. In this case, two biotinylated oligonucleotide probes hybridized with complementary target DNA to create a sandwich hybrid as described above. The DNA hybrids were captured at the surface of the QDs through streptavidin-biotin binding, forming complexes with the two-color cross-linked QDs [38]. Gene expression analysis Recently, a single-molecule approach has been proposed for the direct visualization and mapping of protein binding sites on DNA using QDs. As a proof of concept, this technique was used to identify the genomic targets of transcription

(green) and Cy5 (red) originating a fluorescence image with blended colors in yellow and orange (scale bar 10 µm). (Reprinted with permission from Macmillan Publishers Ltd (Nature Materials) [37], copyright 2005)

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factors (TFs) [39]. Proteins were first cross-linked to DNA and then these complexes were labeled with QDs functionalized with antibodies against the TFs of interest, and the DNA was labeled with an intercalating dye. Owing to the multiplexing capabilities of QDs, different TFs labeled with different QDs allowed multicolor localization with a single excitation source that also excited the DNA stain. This potential to determine the precise loci and occupancy of multiple proteins on long stretches of DNA may elicit interesting applications and give insights into processes such as regulation of gene expression.

M.F. Frasco, N. Chaniotakis

cence detection method for total bacterial count [44]. One procedure combining in vivo biotinylation of an engineered host-specific bacteriophage and conjugation of the phage with streptavidin-coated QDs has been developed. The lysis of infected bacteria in a short time after infection resulted in the release of phages (10–1,000) that could be readily detected by QDs. This method was shown to be rapid, sensitive, and thus particularly useful to detect slow growing or highly infectious bacterial strains susceptible to specific phages [45]. Biosensing of drugs

Detection of proteins and enzymatic activities In a recent work, a nickel–nitrilotriacetic acid (Ni-NTA)– QD cluster was designed to improve the important process of localizing and isolating histidine-tagged fusion proteins [40]. The histidine tag in the recombinant protein serves as a high-affinity binding sequence for the Ni-NTA moiety, which acts as a metal-chelating adsorbent. This technique has been extensively used for purification, immobilization, and detection of recombinant proteins. Therefore, improved intracellular localization and study of protein interactions can be attained by functionalizing the surface of QDs with Ni-NTA. For example, the cluster can exhibit quantitative binding and stability owing to high affinity, site specificity, and reversibility, and one label can be used for multiple targets [40]. There is also great interest in using QDs to develop sensitive sensing approaches for important groups of enzymes (e.g., proteases) that play key roles in normal and pathogenic biochemical processes. Caspases, essential for apoptosis, constitute an example of important clinical and pharmaceutical targets. Recently, the fluorescent protein mCherry was modified to express a target peptide sequence with a caspase 3 cleavage site and a histidine tag to be self-assembled on DHLA-modified QDs [41]. This QD–fluorescent protein conjugate was used to detect the presence of caspase, which cleaved the linker and thus reduced FRET efficiency between the QD and the mCherry acceptor. Pathogen detection QD–antibody or QD–aptamer conjugates capable of binding bacterial surfaces or spores may be very useful in the development of new sensing assays to detect a wide range of harmful biological agents [42, 43]. Other simple modifications of the surface of QDs, for example, with thioglycolic acid, allowed a subsequent covalent conjugation (making use of carbodiimide chemistry) of QDs with target bacteria (Escherichia coli and Staphylococcus aureus) and the development of a sensitive and rapid fluores-

Functionalizing the surface of QDs with aptamers allows for the specific recognition of molecules of interest as diverse as DNA, proteins, and drugs. Recently, an aptameric nanosensor for cocaine was presented, based on single-molecule detection and a bi-FRET mechanism between the QD, fluorophore, and quencher [46]. The cocaine aptamer was sandwiched by a biotinylated oligonucleotide (labeled with a fluorophore at the other end) and a quencher-labeled oligonucleotide. This sandwich hybrid was then assembled on a streptavidin-modified QD. The biFRET mechanism occurred from the QD to the fluorophore and then the fluorophore acted as a donor to the quencher. When cocaine was present, it formed a complex with the aptamer, inducing the release of the quencher-labeled oligonucleotide, and subsequently the fluorescence of the fluorophore was activated. Development of QD-based solid-phase nanosensors Although the vast majority of QD-based nanosensors operate in bulk solution, there is great interest in designing reusable systems. One such example describes the immobilization of QDs on a silica interface by self-assembly using multidentate surface ligand exchange (based on thiolmetal affinity interactions). In this way, QDs retained their ability to interact with target molecules in solution as demonstrated for QD-oligonucleotide and QD–avidin conjugates [47]. QD-FRET-based transduction of nucleic acid hybridization has also been explored in the solid state by immobilizing QD probe–oligonucleotide conjugates on optical fibers [48]. The selective detection of the target occurred upon hybridization of the probe with dye-labeled target oligonucleotides, generating a FRET emission signal as energy was transferred from the QD donor to the dye acceptor. This reusable system exploring solid-phase specific detection of nucleic acids was further enhanced for multiplex analysis of two target sequences and for single nucleotide polymorphism discrimination [49]. It has also been demonstrated that cowpea mosaic virus with specifically designed surface functionalities can be immo-

Bioconjugated quantum dots as fluorescent probes for bioanalytical applications

bilized onto NeutrAvidin-functionalized substrates [50]. This immobilization was accomplished via either biotin– avidin or metal–histidine affinity coordination interactions. The virus particles were then decorated with QDs using a biotin–avidin interaction or a combination of avidin–biotin and metal-affinity coordination. The cowpea mosaic virus has a large surface area and a single virus particle can accommodate several QDs and QD conjugates. This advantage was demonstrated by the larger QD fluorescent signal when QDs were conjugated with the immobilized virus particles than that generated by QDs immobilized on a similar “flat” surface area. Thus, this approach can provide a scaffold for immobilizing an array of molecular assemblies with different functionalities and multiplexing capabilities. QDs incorporated in molecularly imprinted polymers Less explored but already exemplified for the detection of various analytes (e.g., caffeine, uric acid, estriol) is the use of QDs in molecular imprinting, since analyte binding to receptor sites of the host polymer was shown to significantly affect the intensity of QD photoluminescence emission [51]. More recently, with use of the concept of molecularly imprinted nanoparticles, guanosine has been imprinted in the nanoshell of methacryloylamidocysteineactivated CdS QDs using methacryloylamidohistidine platinum as the metal-chelating monomer for DNA recognition via guanosine or its analogues [52]. Bioluminescent QDs QDs as BRET acceptors have been applied in nucleic acid hybridization assays by using Renilla luciferase and QDs labeled with complementary oligonucleotide probes [53]. In the absence of target DNA, the chains hybridized, bringing QDs and luciferase into close proximity, which resulted in an efficient BRET signal. The emission intensity decreased when the target DNA competed to hybridize with the QD probe. BRET-based QD biosensors have also been designed for detecting the activity of matrix metalloproteinases (MMPs) [54]. Here, a luciferase fusion protein with a short peptide containing a MMP substrate and a histidine tag was able to form complexes with carboxylic acid modified QDs through the histidine tag in the presence of nickel ions. BRET was disrupted when the target MMP cleaved the substrate linker, releasing the luciferase from the QDs. These QD-BRET conjugates with demonstrated superior sensitivity and potential for deep tissue imaging lacked stability for long-term imaging studies such as in vivo cell tracking, but this was improved through polymeric encapsulation with a polyacrylamide gel [55].

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Biolabeling and imaging Cell-surface targets Some examples of applications of QDs in tracking and imaging include the specific visualization of serotonin transporter protein [56], G-protein-coupled receptors [57], and GABAc receptors [58]. One such example described the individual tracking of glycine receptors (GlyR), the main inhibitory neurotransmitter receptor in the adult spinal cord, and analyzed their rapid lateral dynamics in the neuronal membrane of living cells for better understanding of the development and plasticity of synapses [59]. The mobility of individual molecules at the neuronal surface was possible owing to the properties exhibited by QDs. Their high photostability also allowed the tracking in the same neuritic region for long durations, in contrast to what is usually accomplished with organic fluorophores. Another example was given on the attachment of QDs to living neurons using both antibody and peptide recognition molecules [60]. With use of this strategy, the targeting and separation distance between the QD and the cell is controlled at the nanometer scale. This property, together with the ability to interface living neurons without the requirement of directed neuronal growth, is very promising for developing bioelectronic devices. The potential of hybrid conjugates of QDs as probes for cell-surface receptors together with the multiplexing capabilities of QDs has been illustrated in multicolor immunophenotyping experiments [61]. Immune cell populations express specific combinations of multiple cell-surface proteins showing enormous heterogeneity. These complex phenotypic patterns can be greatly highlighted by making use of the unique properties of QDs for simultaneous and independent measurements of many proteins, revealing their involvement in immunity and pathogenesis. A general method to target QDs to cell-surface proteins has been proposed. This system involves biotin ligase, which specifically biotinylates any protein bearing an acceptor peptide, which in turn would be recognized by streptavidin-conjugated QDs [62]. Another covalent strategy has been used with the enzyme cutinase, which was fused to target proteins and labeled with QDs functionalized with a suicide inhibitor of the enzyme, which created a covalent linkage between an active-site residue of cutinase and the QD [63]. To avoid the use of large size protein linkers and to increase the prospects of application in living cells, in a recent technique the covalent labeling of cell-surface proteins was based on phosphopantetheinyl transferases. The enzyme catalyzes the transfer of coenzyme A functionalized QDs to a specific serine residue within a short peptide tag fused to the target proteins [64].

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Cell invasiveness

Another interesting work designed lipid-conjugated QDs to be used as single-molecule tracking probes in plasma membranes of several cell types to characterize membrane heterogeneities. The conjugation was achieved by reacting maleimide-containing cross-linker molecules on the surface of QDs with sulfhydryl lipids. The QD–lipid nanocomplex was applied to high-speed single-molecule imaging by tracking the lateral mobility in plasma membranes [65]. Long-term single-QD tracking has also been used to address the nature and dynamics of lipid raft microdomains (cholesterol- and glycosphingolipid-enriched domains in cell membranes that seem to be implicated in signal transduction, amplification, and protein sorting) in the membrane of living cells [66].

An in vitro study of cell invasiveness has been developed using QDs. Motility and migration are characteristics classically associated with tumor cells that invade surrounding tissue and metastasize to different sites in the body [70]. Cells are able to engulf QDs as they move, leaving a phagokinetic track free of QDs and thus it is no longer fluorescent. This sensitive assay was used to compare the behavior of different cell lines, and from the size and shape of the trails cell lines were correlated with the potential invasiveness of those cells. As the imaging of tracks can be carried out on live cells, this assay is a powerful new tool to discriminate noninvasive from invasive cancer cell lines.

QDs targeted to subcellular compartments

Cell signaling pathways

QDs have been used to label different types of target at subcellular locations by using both antibody- and peptidemodified QDs for specific recognition (e.g., intracellular components, intranuclear antigens). The selective targeting of peptide-coated QDs to the vasculature of normal lungs and tumors illustrates the feasibility of in vivo targeting [67]. In this study, the use of poly(ethylene glycol) in the coating of the QDs was shown to prevent the nonselective accumulation of the QDs in reticuloendothelial tissues, which would hinder the successful delivery of the QDs to the target specific tissues and cell types. Another study demonstrated the multiplex ability of QDs by using two QDs with different emission spectra to label the cancer marker HER2 (the human epidermal growth factor receptor 2) simultaneously and specifically at different subcellular levels [68]. QDs linked to IgG were targeted to stain actin and microtubule fibers in the cytoplasm, whereas streptavidinconjugated QDs were used to detect nuclear antigens. Thus, QDs conjugated with different secondary reagents can be effective in fluorescent double labeling. Making use of stability of QDs for potential long-term imaging of live cells, some strategies proved that cells labeled with QDs could be imaged during growth and differentiation [69]. In a first approach, a biotinylated primary antibody was incubated with cells and then an avidin-conjugated QD was used for labeling. In a second method, the primary antibody was first conjugated with the QDs by either a recombinant protein G or avidin and only afterwards incubated with cells. In both approaches, antibodies were electrostatically self-assembled to DHLA-capped QDs. These QD bioconjugates have already been proved to have high specificity and stability in immunoassays, and in this study they enabled specific labeling of proteins for imaging of live cells through noninvasive procedures [69].

The in vivo measurements using QDs have provided new insights into processes and molecular interactions as demonstrated by the use of QDs coupled to growth-factor ligands to study endocytosis [71]. Epidermal growth factor– QD conjugates were able to specifically bind and activate epidermal growth factor receptors (EGFR, erbB1). This caused the receptors to form dimers and thus induce the uptake of the resulting QD–EGFR complex into internal vesicles, or endosomes. Numerous dynamic processes could then be visualized in real time and QDs proved valuable in the study of new signaling cascades.

Diagnosis of metabolic processes Peptide-coated QDs labeled with a fluorescent dye as a molecular acceptor are used to determine enzymatic activities in solution and are the starting point towards a broader application by targeting cells expressing these proteins. A good example is the QD-based FRET sensor for determining proteases (e.g., trypsin, collagenase) by using rhodamine-labeled peptide-coated QDs [72, 73]. For example, the successful quantification of collagenase (a MMP) activity prompted the application of this FRET probe to monitor the proteolytic activity of MMPs in normal and cancerous cell cultures. This real-time discrimination between both types of tissue motivates its use in applications involving screening of overexpression of proteolytic activity [72]. An interesting principle has been applied in the development of an intracellular redox sensor based on QD–dopamine conjugates [74]. Electron donors such as dopamine can act as an electron shuttle between the QDs and other molecules. Conjugation with QDs leads to changes in their optical properties, including photoenhancement and photobleaching. The fluorescence of QDs was

Bioconjugated quantum dots as fluorescent probes for bioanalytical applications

quenched upon conjugation with dopamine, but the complex was able to bind to receptors, ensuring endocytosis. In the intracellular medium, at normal redox potential the endosomes incorporating QD conjugates were only fluorescent after prolonged illumination. In contrast, in highly oxidizing areas the conjugate was impaired and fluorescence was immediately observed without photoenhancement, and in some other regions a slow photoenhancement was observed. A recent FRET study used Nile blue covalently linked to BSA-modified QDs as a fluorescent probe to follow intracellular metabolic pathways, namely, NAD+-dependent biotransformations [75]. This study was further extended by incorporating the designed NADH-sensing QDs into cancer cells to monitor the effect of anticancer drugs on the cell metabolism. When cells were treated with the anticancer drug taxol, the metabolism was suppressed, leading to inefficient yields of NADH, which were sensed by lower fluorescence intensities of the QDs. Therefore, besides the very broad applicability of the functionalized QDs to probe a wide class of enzymes, QDs can also be used for screening anticancer drugs that affect the intracellular metabolism.

Intracellular delivery of therapeutic agents An insightful overview of the various strategies currently in use for delivery of QDs into cells, their advantages and liabilities, as well as major determinants for efficient cellular uptake has recently been provided [76]. Here, we provide some examples of QD-mediated simultaneous imaging and delivery of agents with therapeutic potential. QDs have been used as fluorescent probes to track the binding, cell surface motion, and internalization via endocytosis of vectors for gene delivery [77]. In search for improved gene, protein, or other molecular cargo delivery systems, cationic polymers and peptides such as polyethyleneimine and polyarginine have gained increasing importance. Cationic ligand–QD complexes were applied to study the overall dynamics of the process, linking motion to delivery of genes to cells. Some strategies for developing QD–cell-penetrating peptide bioconjugates for intracellular delivery have been attempted. A bifunctional oligoarginine cell-penetrating peptide bearing a polyhistidine sequence for self-assembly onto the surface of QDs was shown to facilitate the transmembrane delivery of the QD bioconjugates and intracellular labeling [78]. One of the interesting aspects of using QDs functionalized with cell-penetrating peptides is the possibility to expand the technique to promote the intracellular delivery of QDs decorated with small or large protein cargos [79]. Proteins such as yellow fluorescent

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protein or the multichromophore b-phycoerythrin complex were conjugated with QDs functionalized with cellpenetrating peptides. The intracellular delivery of these bioconjugates was shown to be dependent on the cellpenetrating peptide. Thus, the QD surface engineered with therapeutic agents can make the nanoassembly function simultaneously for imaging and drug delivery. RNA aptamers covalently attached to the surface of QDs have been used as targeting molecules and as specific vectors for intracellular delivery of therapeutic agents to prostate cancer cells. QD–RNA aptamer recognizing prostate-specific membrane antigen (PSMA) was conjugated with doxorubicin, an antineoplastic anthracycline drug with fluorescent properties. The delivery of this drug to the targeted tumor cells was monitored on the basis of a biFRET mechanism. The intercalation of doxorubicin within the PSMA aptamer resulted in quenching of both QD and drug fluorescence since QDs function as FRET donors to doxorubicin, which in turn intervenes in the donor– quencher FRET model with the aptamer [80]. Upon PSMA-mediated endocytosis, the drug was released and the recovery of fluorescence from both QDs and doxorubicin was achieved, enabling the synchronous fluorescent localization and intracellular drug delivery for destroying cancer cells (Fig. 5a). Another such application with great therapeutic potential has been reported on the basis of an intrinsic biological mechanism. RNA interference is a pathway within living cells that controls gene expression, whereby short stretches of double-stranded RNA with sequences complementary to a gene sequence suppress the activity of the gene by degrading the corresponding messenger RNA and thus blocking its translation into proteins [81, 82]. This is a valuable research tool since synthetic short interfering double-stranded RNA can be codelivered with fluorescent QDs into cells using standard transfection techniques inducing the suppression of specific genes of interest [81]. For instance, QDs encapsulated in chitosan nanoparticles were used to deliver the HER2 short interfering RNA (siRNA). The complexes were successfully tracked and monitored in vitro, with the internalization into cells accomplishing the silencing effects on the HER2 gene [82]. An improved approach for a targeted and systemic delivery of siRNA relied on the conjugation of siRNA and tumor-homing peptides with functional groups on the surface of PEGylated QDs [83]. Moreover, the addition of endosome-disrupting agents was required to aid the release of siRNA from the endosomal entrapment, enhancing the knockdown efficacy. The advantageous features of this multifunctional complex can thus be explored by designing siRNA sequences against different therapeutic targets for synchronized treatment and imaging applications (Fig. 5b).

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a

b

QD-bioconjugates for intracellular delivery of therapeutic agents

Specific recognition at the target cell membrane and receptor-mediated endocytosis

Fluorescence imaging and delivery of cargo-molecules for therapeutic purposes

Fig. 5 Synchronous imaging and delivery of therapeutic agents to target cells. a QD–aptamer conjugates provide recognition by specific membrane receptors that mediate endocytosis and function as drug carrying vehicles (e.g., bi-FRET mechanism designed for imaging and delivery of an antineoplastic drug to targeted tumor cells [80]). b Cell

internalization of QD–short interfering RNA (siRNA) conjugates mediated by targeted peptides also attached to functional groups on the surface of QDs for RNA-induced gene silencing (e.g., significant knockdown of the gene of interest achieved after targeted internalization by tumor cells [83])

Conclusions and outlook

Cd2+ ions, causing cell death) [85]. A direct way to avoid the possible toxicity of QDs is to coat them with biologically inert layers as previously discussed, reducing the possible leaking of toxic metals in physiological environments. However, it has also been reported that the formation of aggregates on the cell surface and the stability of the surface capping/cytotoxicity of surface ligands are factors that may contribute to impairing cell viability [86, 87]. Other aspects such as possible nonspecific binding to cellular membranes and intracellular proteins, formation of reactive oxygen species, and genotoxicity are certainly of concern [88]. It has been reported that QDs are taken up nonspecifically by the reticuloendothelial system, including the liver and spleen, and the lymphatic system, where their fluorescence could be detected for long periods of time, indicating long-term stability without degradation into their potentially toxic elemental components [89]. The entrapment of QDs in the reticuloendothelial system certainly has toxicological implications since it is a defensive mechanism of the organism, but also the applicability of QDs as fluorescent probes may be compromised if they are not able to reach their target sites. It has been suggested that the size of the QDs, their surface ligands and coatings, including the overall charge influence the lifetime of QDs in circulation, tissue deposition, and hepatic or renal clearance [90, 91]. Thus, it will be crucial to pursue studies of the cellular toxicity, tissue and organ clearance, and in vivo degradation mechanisms of QD probes. Moreover, together with the development of stable and robust coating strategies to minimize the toxicity of currently available QDs, progress should be directed to develop new high-quality QDs of more biocompatible materials. Looking to the future of QDs technology, there is a great bioanalytical potential and as soon as these limiting issues have been addressed, the field will take a fairly substantial leap forward.

QDs have revolutionized the field of biological sensing and labeling. Improvements in the synthesis and functionalization of QDs with targeting ligands such as nucleic acids, peptides, antibodies, and small molecules are continuing. The specificity achieved together with the sensitivity of the fluorescent signal encouraged the integration of QDs in a myriad of nanosensors. The bioanalytical applications of these QD bioconjugates such as noninvasive targeting, imaging, and delivery of therapeutic agents are thus very auspicious. Though QDs are highly fluorescent and photostable, the size compatibility issue is still unresolved. By their colloidal nature, QDs are substantially larger that molecular dyes and the nanocrystal radius increases for redemitting QDs, which are more suitable for in vivo studies. In addition, the hydrophilic QDs used in biological applications attain their lower toxicity, higher stability and quantum yield, and specificity to targets through ligand exchange or encapsulation in biocompatible matrices. These surface-functionalization procedures can dramatically increase the overall nanoconjugate size, limiting their tissue penetration. In addition, routine in vivo labeling and imaging assays have been precluded so far owing to the toxicological hazard of QDs. This toxicity is usually connected to the central metal core of QDs, and the pharmacology and toxicology of the most commonly used cadmium chalcogenide based QDs have been recently reviewed [84]. Although the use of QDs in living cells and animals normally includes surrounding the nanocrystalline core with biologically inert ZnS and encapsulation within stable matrices, the actual impact of in vivo use of QDs is still unclear. The cytotoxicity of CdSe core QDs towards hepatic primary cells has been demonstrated in vitro under UV irradiation (surface oxidation led to the release of free

Bioconjugated quantum dots as fluorescent probes for bioanalytical applications Acknowledgements Financial support by Fundação para a Ciência e a Tecnologia (M.F.F., postdoctoral grant SFRH/BPD/40876/2007), and by the European Commission program NANOMYC (contract 036812) is gratefully acknowledged.

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