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Integrated BiomoleculeQuantum Dot Hybrid Systems for Bioanalytical Applications Ronit Freeman, Bilha Willner, and Itamar Willner* Institute of Chemistry, Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel ABSTRACT: Recent scientific efforts are directed to the coupling of biomolecules with semiconductor quantum dots (QDs) to yield hybrid nanomaterials. The biomolecule/QDs conjugates combine the unique optical and electrical properties of QDs with the recognition and catalytic functions of biomolecules and provide new materials for versatile bioanalytical applications. The article addresses different approaches that implement functional biomoleculeQD hybrid systems for sensing applications. QDs are implemented as fluorescent labels for biorecognition events, and the size-controlled luminescence features of QDs are used for the development of multiplexed analysis schemes. Also, fluorescence resonance energy transfer (FRET), electron transfer, and chemiluminescence resonance energy transfer (CRET) processes are used to probe the dynamics of biorecognition events and biocatalyzed transformations. Specifically, the incorporation of functional QDs into cells holds great promise for monitoring intracellular metabolic pathways and for future applications in nanomedicine. Finally, photocurrents stimulated by QDs linked to electrodes are used to transduce biorecognition events and biocatalytic processes.
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emiconductor quantum dots (QDs) exhibit unique sizecontrolled photophysical properties reflected by high luminescence intensities and quantum yields, size-controlled absorption and luminescence features, large stokes shifts, and stability against photobleaching.14 Biomolecules, in turn, reveal evolutionary-optimized specific, high affinity, binding features or biocatalytic functions. The nanometer-sized dimensions of biomolecules and QDs suggest that combining the two components into hybrid systems could yield new materials, where the optical and electronic properties of the QDs are utilized to transduce the functions of biomolecules. Indeed, tremendous progress in the application of QDs for biosensing and imaging has been achieved in recent years, and the topic has been comprehensively reviewed.5,6 In this Perspective we aim to discuss the principles for applying QDs for biosensing and imaging, while discussing several examples and introducing new sensing paradigms. Different methods to prepare QDs are available, including the stabilization of semiconductor nanostructures by protecting monolayers7 or thin films,8 the synthesis of the QDs in microemulsion nanoreactors,9 and more.10 The application of QDs for biosensing requires, however, the preservation of their photophysical properties in aqueous media. Different methods to retain the photophysical properties of the QDs in aqueous environments have been reported, and these include the capping of the QDs with thiolated monolayers,11,12 amphiphilic polymer micelles,13 or peptides14,15 (e.g., glutathione). The capping of the QDs with functional monolayers or thin films not only preserves the properties of the QDs, but it enables the tethering of chemical functionalities to the QDs. These chemically modified QDs hybrid systems may be implemented for bioanalysis using several sensing paradigms (Figure 1). These include the use of QDs as r 2011 American Chemical Society
Figure 1. Schematic applications of semiconductor QDs for the analysis of recognition complexes by FRET, ET, or photocurrent readout mechanisms.
luminescent labels for biorecognition events, the stimulation of fluorescence resonance energy transfer (FRET) or electron transfer (ET) processes as a result of the sensing events, the stimulation of a chemiluminescence resonance energy transfer (CRET) process upon recognition of the analyte, the activation of a photocurrent upon the sensing of the analyte, and implementing the photoelectrochemical functions of the QDs. The multiplexed analysis of antigens using different sized CdSe/ZnS QDs is exemplified in Figure 2A using the different, size-controlled, luminescence properties of the QDs.16 The QDs were functionalized each by antibodies against the toxins cholera Received: May 16, 2011 Accepted: September 23, 2011 Published: September 23, 2011 2667
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Figure 2. (A) Multiplexed luminescence analysis of different toxins on a surface using a “sandwich-type” immuno-complex assay and different-sized CdSe/ZnS QDs. (B) Luminescence spectra (a) and their deconvolution upon simultaneous analysis of the four toxins: (b) choleratoxin, (c) ricin, (d) SLT, and (e) staphylococcal. Reprinted with permission from ref 16. Copyright 2004 American Chemical Society. (C) Analysis of a target DNA by a hairpin nucleic acid probe functionalized with CdSe/ZnS QD and quencher units. (D) Luminescence spectra of the QDs: (a) in the hairpin configuration; (b) upon opening of the hairpin by the target DNA, and upon interaction of the functionalized hairpin with (c) single base mismatched target DNA and (d) non-complementary target DNA. Reprinted with permission from IOP Publisher Ltd. from ref 18. (E) Analysis of cocaine by the self-assembly of the cocaine-aptamer subunits complex and the activation of the FRET process in the complex. The aptamer subunits were modified with CdSe/ZnS QDs and Atto-590 dye, respectively. (F) Time-dependent luminescence spectra corresponding to the dynamics of the self-assembly of the cocaine-aptamer subunits and demonstrating intracomplex FRET process. Reproduced by permission of The Royal Society of Chemistry (RSC) from ref 22.
(CT), ricin, shiga-like toxin 1 (SLT), and staphylococcal enterdoxin B (SEB). A single well of microtiter plate was modified with the antibodies against all antigens. The multiplexed analysis of the antigens was demonstrated by a “sandwich-type” immunoassay, where the formation of the respective immunocomplexes was imaged by analyzing the luminescence spectra generated by the different QDs labels (Figure 2B). The use of luminescent QDs for the multiplexed imaging of biorecognition events was, similarly, applied to analyze different DNAs such as the detection of single-nucleotide polymorphisms of the human oncogene p53 or the multiallele detection of hepatitis B and C.17 FRET has been used to develop numerous sensing platforms. Since the FRET process sharply depends on the distance separating the donoracceptor pair, recognition events that control the distances between the donor and acceptor units are ideal to be probed by the FRET process. Figure 2C outlines the detection of DNA by a nucleic acid hairpin probe functionalized
at the end of the stem region with a QD (donor) and the BHQ-2 dye (acceptor), (a molecular beacon).18 The proximity between the QDs and the acceptor dyes leads to the quenching of the luminescence of the QDs. The hybridization of the target DNA with the loop region of the hairpin structure opens the molecular beacon, leading to the spatial separation of the QDs from the quencher units. This triggers-on the luminescence of the QDs (Figure 2D). Similarly, the FRET process was used to develop aptamer-based sensors. Aptamers are nucleic acids exhibiting specific recognition properties for low-molecular-weight substrates or macromolecules. The aptamers are prepared by a selection and amplification protocol, the systematic evolution of ligands by exponential enrichment, SELEX process,19,20 and they gain substantial interest as selective binding sites for bioanalytical applications.21 Figure 2E depicts the analysis of cocaine by a FRET-based aptasensor.22 The anticocaine aptamer was fragmented into two subunits (1 and 2), where the 30 -end of 1 was tethered to CdSe/ZnS QDs, and the 50 of the subunit 2 2668
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Figure 3. (A) Analysis of a target DNA by hairpin-functionalized QDs. Opening of the hairpin by the analyte leads to the self-organization of the hemin/ G-quadruplex that queches the QDs via an electron transfer process. Reprinted with permission from ref 27. Copyright 2006 American Chemical Society. (B) Multiplexed analysis of three different target DNAs using different sized CdSe/ZnS QDs, each modified by a specific hairpin structure for sensing each of the analytes. Opening of the hairpin structure by the respective target results in the self-assembly of the hemin/G-quadruplex on the respective QDs, and this activates the CRET to the QDs, resulting in the triggered-on luminescence of the different semiconductor nanoparticles: (a) QDs emitting at λ = 620 nm. (b) QDs emitting at λ = 560 nm. (c) QDs emitting at λ = 490 nm. (C) CRET signal of the 620 nm-QDs upon analyzing different concentrations of the target DNA: (1) no target, (2) 10 nM, (3) 25 nM, (4) 50 nM, (5) 100 nM. (D) CRET signals of the different sized QDs upon the multiplexed analysis of the different target DNAs: (1) in the absence of the different DNA targets; (2) in the presence of the respective target for the hairpin-modified 560 nm-QDs; (3) in the presence of the respective target for the hairpin-modified 620 nm-QDs; (4) in the presence of the respective target for the hairpin-modified 490 nm-QDs; (5) in the presence of all three different DNA targets. (BD) Reprinted with permission from ref 29. Copyright 2011 American Chemical Society.
was modified with the atto-590 fluorophore. Although the subunits 1 and 2 include partial complementarity, the resulting base-pairing is insufficient to stabilize the formation of the intact aptamer structure. In the presence of cocaine, the complex between the analyte and the aptamer subunits is cooperatively stabilized by the duplexes between the subunits and their interaction with cocaine, leading to the supramolecular cocaine aptamer subunits complex. The close proximity between the QDs and the fluorophore yields a FRET process. The timedependent assembly of the supramolecular complex was followed by the FRET signal (Figure 2F). By monitoring the FRET signal intensity in the presence of different concentrations of cocaine and at fixed time-intervals of assembly of the aptamercocaine complex, the cocaine was analyzed with a detection limit corresponding to 1 106 M. The electron transfer quenching of QDs was also implemented to follow recognition events. The hemin/G-quadruplex was found to act as a horseradish peroxidase-mimicking catalytic nucleic acid (DNAzyme), 23 and it was used as a catalytic amplifying label in numerous bioanalytical platforms. 2426
It was found, however, that the hemin/G-quadruplex linked to CdSe/ZnS QDs acts as an effective electron transfer quencher of the luminescence of the QDs. This feature was used to develop different DNA or aptamer-based sensing platforms.27 Figure 3A depicts the analysis of DNA by the electron transfer quenching mechanism of the QDs. The hairpin, 3, included in its stem region the G-quadruplex sequence in a “blocked” configuration (region I) and a single-stranded recognition loop domain (region II). Upon hybridization of the analyte DNA with the loop, the hairpin was opened, resulting in the self-assembly of the hemin/G-quadruplex units that quenched the QDs. The hemin/G-quadruplex nanostructure was also implemented for the CRET detection of aptamer-substrate complexes and of DNA, and the multiplexed analysis of DNAs was demonstrated with this analytical platform. The hemin/G-quadruplex acts as a catalyst for the generation of chemiluminescence (λem = 420 nm) in the presence of luminol and H2O2.28,29 The CRET process proceeds at short distances separating the chemiluminescence source and the energy acceptor units. Accordingly, hemin/G-quadruplex-functionalized semiconductor QDs were used as hybrid nanostructures for bioanalytical 2669
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Figure 4. (A) Probing the collagenase activity by following the cleavage of the dye-modified sequence-specific peptide linked to the QDs. The FRET process between the QDs and the dye is blocked upon the cleavage of the peptide, resulting in the enhanced luminescence of the QDs. (B) Luminescence spectra corresponding to the cleavage of the peptide by different concentrations of collagenase. Reprinted with permission from ref 30. Copyright 2006 American Chemical Society. (C) (I) Probing CK activity through the phosphorylation of the sequence-specific peptide and the labeling of the phosphate group with Atto-590, resulting in a FRET signal that follows the catalytic transformation. (II) Structure of the dye-labeled ATP. (III) Time-dependent luminescence spectra following the dynamics of the phosphorylation process by the FRET mechanism. Reprinted with permission from ref 32. Copyright 2010 American Chemical Society. (D) Probing the HAT activity using a substrate peptide-modified QDs, acetyl-CoA, and dye-labeled antiacetyl antibodies. (E). Time-dependent luminescence spectra following the dynamics of the acetylation process by the FRET mechanism taken at 15 min intervals. Reproduced with permission from ref 34.
applications, using the DNAzyme-generated chemiluminescence as an internal light source for the detection of DNA, and using the luminescence of the QDs as a readout signal (Figure 3B). CdSe/ZnS QDs (λem = 620 nm) were functionalized with a hairpin nucleic acid nanostructure that included a “blocked” G-quadruplex sequence in the stem region of the hairpin, while the single-stranded domain provided the recognition site (Figure 3B,a). In the presence of the target DNA, the hairpin structure opened, leading to the self-assembly of the hemin/ G-quadruplex. The DNAzyme-generated chemiluminescence, λ = 420 nm, stimulated the CRET to the QDs, a process that activated the luminescence of the QDs (Figure 3C). This method enabled the analysis of DNA with a detection limit corresponding to 10 nM. The potential of the CRET-based nucleic acid-functionalized QDs in bioanalysis rests, however, in the easy adaptation of the method and applynig different sized hybrid QDs for multiplexed sensing. The possibility to excite different sized CdSe/ZnS QDs by the chemiluminescence light source suggests that multiplexed detection of different target nucleic acids could be achieved by the selective activation of the luminescence of the different sized QDs, dictated by the selective sensing process
(Figure 3B). Three different sized QDs were modified with three different hairpin nucleic acid probes. All three nucleic acids included in their stem region the “caged” G-quadruplex structures, but their single stranded loop domains were different and were designed to be complementary to three different DNA targets. By the selective opening of each of the hairpin-modified QDs, the luminescence of the respective QDs was triggered on, and in the presence of all three target DNAs, the luminescence of all different sized QDs was switched on (Figure 3D). This suggests that the complexity of parallel analysis of targets might be enhanced by adding different sized QDs of other materials, and different shaped QDs exhibiting tunable luminescence features.
Hemin/G-quadruplex-functionalized QDs act as hybrid nanostructures for bioanalytical applications. 2670
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Figure 5. (A) Analysis of M13 phage DNA by nucleic acid-functionalized QDs. The polymerase-stimulated replication of the duplex generated between the probe nucleic acid and the target, in the presence of polymerase, dNTPs and Texas-Red-labeled dUTP, leads to a FRET process between the QDs and the labeled replica. (B) Time-dependent FRET spectra following the replication process (a) before addition of Texas-Red-labeled-dUTP and (bd) after 1, 30, and 60 min of replication. Reprinted with permission from ref 35. Copyright 2003 American Chemical Society. (C) Analysis of tyrosinase activity by the biocatalytic oxidation of a methyl ester tyrosine-modified CdSe/ZnS QDs. The biocatalytic oxidation of the capping layer to dopaquinone leads to an electron transfer quenching of the QDs. (D) Time-dependent luminescence quenching of the QDs upon the biocatalytic oxidation of the tyrosine substrate for (a) 0 min, (b) 0.5 min, (c) 2 min, (d) 5 min, and (e) 10 min. Reprinted with permission from ref 40. Copyright 2006 American Chemical Society.
Probing Dynamic Transformations with QDs. The intimate distance dependency of the FRET process between QDs and dyes provides a general principle that can be applied to follow the dynamics of biocatalytic transformations. Specifically, the enzymatic cleavage of biomolecular substrates labeled with a QD/dye pair, or the biocatalyzed build-up of the QD/dye pair on a QD functionalized with the biomolecular substrate, may be followed by the FRET process. For example, different proteolytic enzymes, e.g., collagenase (Figure 4A), were probed using the FRET process.30,31 The CdSe/ZnS QDs were functionalized with peptides modified at their ends with the Rhodamine Red-X acceptor. An effective FRET process between the QDs and the dye units occurred in the hybrid nanostructure. The sequencespecific cleavage of the peptide that acted as a substrate for collagenase separated the dye from the particle, resulting in a decrease of the FRET signal and an increase in the luminescence of the QDs (Figure 4B). The build-up of the FRET signal as a result of a biocatalytic transformation is exemplified with the detection of casein kinase (CK), a general biomarker for cancer cells.32 CK is a serine phosphorylation biocatalyst. Accordingly, CdSe/ZnS QDs were functionalized with the serine-containing peptide, and it was reacted with atto-590-labeled adenosine triphosphate (ATP) in the presence of CK to yield the dyelabeled phosphorylated peptide (Figure 4C). The FRET process from the QDs to the dye units (Figure 4C,III), enabled to follow the activity of CK. Using a related approach, the antibody that binds specifically to the phosphorylated peptide was modified with the atto-590 dye. The association of the antibody with the phosphorylated product stimulated the FRET process from the QDs to the dye units, and the FRET signal generated by the atto590 dye enabled the detection of CK. Other protein kinases were similarly monitored using a FRET process between QDs and
dye-labeled antibodies associated with the phosphorylated product.33 This concept was further extended to monitor other enzyme-catalyzed transformations on peptide residues. For example, histone acetyl transferases (HATs) are associated with numerous pathological conditions, such as, cancer, neurodegeneration, and HIV infection, and they catalyze the acetylation of lysine residues using acetyl-CoA as the acetyl function source. Accordingly, CdSe/ZnS QDs were functionalized with the lysine-containing histone tail, and the QDs conjugates were reacted with HAT p300 and acetyl CoA (Figure 4D). The selective association of the dye-functionalized antibody to the acetylated peptide resulted in a FRET process between the QDs and the dye units (Figure 4E), thus enabling the quantitative assay of HAT.34
QDs follow the dynamics of biocatalytic transformations. The FRET reaction was also implemented to follow biocatalytic transformations on DNA substrates, such as replication,35 telomerization,35 or scission.36 For example, CdSe/ZnS QDs were functionalized with the nucleic acid 5, which is complementary to a domain of the M13 phage DNA. Hybridization of the target M13 phage DNA with the probe 5 associated with the QDs was followed by the replication of the resulting hybrid in the presence of polymerase and the nucleotides mixture, dNTPs, that included Texas-Red-functionalized dUTP (Figure 5A). The dye incorporated into the replicated strand activated the FRET process from the QDs to the dye (Figure 5B). As the replication 2671
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The Journal of Physical Chemistry Letters proceeded, the content of the Texas-Red dye incorporated in the replicated target associated with the QDs increased, resulting in an intensified FRET signal of the dye at λ = 610 nm.35 A related approach was applied to follow the activity of telomerase in cancer cells. Telomerase is a ribonucleoprotein that elongates the telomer ends associated with the chromosomes, and it is considered as a versatile biomarker for cancer cells.37,38 The detection of telomerase was achieved by subjecting CdSe/ZnS QDs modified with a nucleic acid primer unit, that acts as a substrate for telomerase, to the telomerization process. In the presence of the cancer cell extract and with the added nucleotides mixture (dNTPs), which included the Texas-Red-dUTP, telomerization of the primer nucleic acid (associated with the QDs) occurred, resulting in the incorporation of the dye into the telomer chains. The close proximity between the QDs and the dye stimulated an effective FRET from the QDs to the Texas-Red dyes. The dynamics of the telomerization process was then followed by probing the time-dependent enhancement of the FRET-stimulated fluorescence of the dye.35 A related approach was used to follow the DNase I cleavage of DNA.36 A 50 -thiolated nucleic acid was immobilized on CdSe/ZnS QDs, and the complementary nucleic acid modified at its 30 -termini with Texas-Red was hybridized with nucleic acid-functionalized QDs. The FRET process from the QDs to the dye triggered on the fluorescence of the dye units. The cleavage of the DNA duplex by DNase I separated the dye from the QDs, a process that regenerated the luminescence of the QDs due to the elimination of the FRET process in the hybrid. Electron transfer quenching of the luminescence of the QDs was also used to probe the dynamics of biocatalytic processes. Tyrosinase catalyzes the oxidation of phenolic substrates to ortho-biphenolic products, which are further oxidized to ortho-quinone derivatives. Elevated amounts of the enzyme are present in melanoma cancer cells, and it is considered as a biomarker for this type of cells.39 The activity of tyrosinase was monitored by CdSe/ZnS QDs, functionalized with tyrosine methyl ester residues40 (Figure 5C). The tyrosinase-stimulated oxidation of the tyrosine substrate to the respective L-DOPA and, subsequently, to the quinone residues led to the electron transfer quenching of the luminescence of the QDs. The time-dependent quenching of the QDs provided a pathway to follow the dynamics of the biocatalytic oxidation of the substrate (Figure 5D), and by probing the luminescence of the QDs after a fixed time-interval of the biocatalytic process, variable concentrations of the enzyme could be quantitatively assayed. Probing Intracellular Processes with QDs. Different methods to incorporate QDs into cells were developed. These include nonspecific endocytosis,41 the use of receptor-functionalized-QDs as transfection reagents,42,43 and the application of dendrimermodified or polymer-functionalized QDs.44 Also, physical techniques such as microinjection or electroporation were employed for the delivery of QDs into cells.45,46 The QDs incorporated into cells have been extensively implemented for the labeling and imaging of subcellular compartments.47 The great promise of functionalized QDs rests, however, in their potential to optically image intracellular processes, and to probe the dynamic transfer and delivery of drugs into cells. While these applications are scarce, recent reports emphasize the future significance of such applications of QDs. The use of QDs as a “carrier” of molecular or macromolecular “cargoes” provides a versatile means to transport therapeutic materials into cells and to optically probe their localized release. This was exemplified with the modification of QDs with the RNA aptamer against the prostate-specific membrane antigen, and the intercalation of the anticancer doxorubicin drug into duplex
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domains of the aptamer units (Figure 6A).48 Electron transfer quenching of the luminescence of the QDs occurred in the resulting hybrid nanostructure. The incorporation of the functionalized QDs into cancer cells stimulated the release of doxorubicin, which killed the cells, while restoring the luminescence of the QDs. A related approach was used for the intracellular delivery of small interfering RNA (siRNA) for different genes, which enabled the synchronized treatment and imaging of the cells.49 The incorporated siRNA hybridized with the messenger RNA (mRNA), and this prevented translation of proteins. Accordingly, different siRNA-functionalized QDs were delivered into cancer cells, and these suppressed different genes, such as the lamin A/C gene or HER2 gene. In addition to the delivery and imaging functions of cellincorporated QDs, the photophysical properties of QDs can be implemented as reporter units for intracellular processes, and particularly, for monitoring cell metabolism. This was exemplified with the application of QDs for monitoring the metabolism in HeLa cancer cells, while probing the effect of anticancer drugs on the intracellular metabolic pathways.50 The CdSe/ZnS QDs were functionalized with a bovine serum albumin (BSA) monolayer, and the redox dye Nile Blue was covalently tethered to the protein layer (Figure 6B). The color of the dye overlaps the luminescence spectrum of the QDs, leading to the FRET quenching of the luminescence of the QDs. The reduced cofactor, 1,4-dihydronicotinamide adenine dinucleotide, NADH, reduces the redox dye to a colorless product, and thus, the FRET process between the QDs and the redox dye is prohibited, and the luminescence of the QDs is triggered-on (Figure 6C). Accordingly, the Nile-Blue-functionalized QDs were applied for the optical sensing of the NADH cofactor, and for the optical detection of the activity of NAD+-dependent enzymes and their substrates.50 A major advance was demonstrated, however, upon applying the modified QDs for monitoring the metabolism in HeLa cancer cells. As the cell metabolism yields NADH, changes in the luminescence of the functionalized QDs incorporated in the cells reflect dynamic alterations of the cell metabolism. The Nile-Blue-modified QDs were incorporated into HeLa cancer cells by electroporation, and the cells were cultured under “starvation” conditions. Upon the addition of D-glucose to the growth medium, the cells metabolism was activated. This process intensified the luminescence of the QDs in the cells as a result of the generation of the NADH cofactor (Figure 6D, curve 1). Interestingly, non-native L-glucose had no effect on the cell metabolism (Figure 6D, curve 2). The optical monitoring of the HeLa cell metabolism was then applied to probe the effect of the anticancer taxol drug on the cell metabolism. The metabolism of taxol-treated HeLa cancer cells was compared to the progress of the metabolism in nontreated cells (Figure 6E). The taxol-treated cells revealed an inefficient activation of the cell metabolism upon the addition of D-glucose, reflected by low luminescence changes in the cells. These results do not only confirm that taxol inhibits the metabolism of cancer cells, but demonstrate the potential application of the modified QDs for screening drugs affecting intracellular metabolic pathways.
The photophysical properties of QDs are implemented as reporters for intracellular processes. 2672
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Figure 6. (A) (I) Binding of doxorubicin to the respective aptamer-functionalized QDs and (II) incorporation of the doxorubicin aptamer-QDs hybrids into cancer cells. The intracellular release of doxorubicin induced an enhancement in the luminescence of the QDs. Reprinted with permission from ref 48. Copyright 2007 American Chemical Society. (B) The application of Nile-Blue-functionalized QDs for the optical detection of the NADH cofactor. The reduction of Nile-Blue by the NADH triggers-on the luminescence of the QDs. (C) Time-dependent increase in the luminescence of the QDs, upon interacting with NADH, 0.5 mM (1) before addition of the NADH and (26) after successive time intervals of 3 min. (D) Time-dependent luminescence change of Nile-Blue-functionalized QDs incorporated into HeLa cancer cells cultured under starvation conditions (1) upon reaction with D-glucose 50 mM and (2) upon interaction with L-glucose 50 mM. Inset: Confocal fluorescence microscopy images of representative HeLa cancer cells before and after the activation of the intracellular metabolism by D-glucose. (E) Time-dependent fluorescence change by Nile-Blue-functionalized CdSe/ ZnS QDs associated with HeLa cancer cells grown under starvation (1) in the absence of taxol (2) in the presence of taxol, and upon the activation of the intracellular metabolism by adding to the different cells 50 mM D-glucose. The low increase in the luminescence of the taxol-treated cancer cells implies that the cell metabolism was inactivated by taxol. (BE) Reproduced with permission from ref 50. (F) Monitoring pH levels using dopaminefunctionalized QDs. As pH increases, the capping layer is oxidized to dopaquinone, resulting in an electron transfer quenching of the QDs. (G) Luminescence intensities of the modified QDs at different pH levels. Inset: The quenching degree of the modified QDs at different pH ranging from 10.1 to 4.8. Reproduced with permission from ref 53. Copyright 2010 Nature Publishing Group.
Also, functionalized QDs were implemented as oxygen sensors using a two-photon excitation process.51 The fact that QDs can be encapsulated in cleavable polymer matrices, such as gelatin, and deliverd into tumor cells,52 suggests that appropiately functionalized QDs could monitor oxygen levels in tumor or inflamed cells, and concomitantly release therapeutic drugs. An interesting method to probe intracellular pH changes was reported using dopamine-functionalized QDs (Figure 6F).53 CdSe/ZnS QDs were modified with peptide chains, to which dopamine was tethered. The dopamine is oxidized by oxygen to form the dopaquinone units exhibiting electron acceptor properties. As the oxidation of dopamine is facilitated as the pH of the medium turns basic, the electron transfer quenching of the QDs was enhanced (Figure 6G). Upon the incorporation of the modified QDs into cells, the luminescence features of the QDs probed effectively intracellular pH changes. Similarly, the dopamine-functionalized CdSe/ZnS QDs were used to probe the oxidative environments of different cell compartments,54 thus demonstrating that the redox-sensitive-modified QDs could probe metabolic
intracellular processes, cell stress phenomena, and antioxidants activities. QDs for Photoelectrochemical Biosensing. Photoexcitation of the QDs leads to the formation of an electronhole pair. This exciton decays to the ground state either via electron transfer quenching by solution solubilized electron acceptor or electron donor, exhibiting appropriate energy levels, or by the radiative electronhole recombination that yields luminescence and is eventually accompanied by the secondary FRET processes. A further pathway for the decay of the exciton generated in the QDs includes a photoelectrochemical mechanism that involves the coupling between the photoexcited QDs and an electrode surface (Figure 7A). The ejection of the conduction-band electrons to the electrode with the concomitant reduction of the valenceband holes by an electron donor leads to the generation of an anodic photocurrent (Figure 7A,I). Alternatively, the ejection of the conduction-band electrons to an electron acceptor solubilized in the electrolyte solution and the transfer of electrons from the electrode to the valence-band holes (Figure 7A,II) yield a cathodic photocurrent. While the photoelectrochemical effect is 2673
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Figure 7. Development of photoelectrochemical biosensing assays using semiconductor QDs: (A) Schematic anodic (I) and cathodic (II) photocurrents generated by QDs-modified electrodes. (B) A photoelectrochemical cocaine sensor based on the formation of a cocaine/aptamer subunits complex on an electrode support. (C) Photocurrent action spectra corresponding to the analysis of different concentrations of cocaine by the functionalized electrode: (a) in the absence of cocaine (b) 1 106 M (c) 1 105 M (d) 1 104 M (e) 1 103 M. Inset: The derived calibration curve. Reprinted with permission from ref 57. Copyright 2009 American Chemical Society. (D) Photoelectrochemical probing of tyrosinase activity by the biocatalytic oxidation of methyl ester tyrosinefunctionalized CdS nanoparticles and the subsequent association of the biocatalytically generated L-DOPA-functionalized nanoparticles to a phenylboronic acid monolayer-modified electrode. Reprinted with permission from ref 58. Copyright 2008 American Chemical Society. (E) Assembly of a CdS NP/AChE hybrid system for the photoelectrochemical detection of the enzyme activity. (F) Photocurrent action spectra observed in the presence of (a) 0, (b) 6, (c) 10, (d) 12, (e) 16, and (f) 30 mM acetylthiocholine (6). Inset: Calibration curve of the photocurrent at λ = 380 nm at various concentrations of 6. (G) Photocurrent spectra for the CdS/AChE system in the presence of 6 (10 mM): (a) in the absence of the inhibitor, (b) upon addition of the inhibitor (1 106 M), and (c) after rinsing the system to remove the inhibitor, and addition of 6 (10 mM). (EG) Reprinted with permission from ref 59. Copyright 2003 American Chemical Society. 2674
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The Journal of Physical Chemistry Letters vastly used in the development of solar cells,55,56 recent research efforts are directed to implement this light-to-electrical energy conversion process for developing optoelectronic biosensors. Two general paradigms are used to develop QDs-based photoelectrochemical biosensors. One approach involves the use of the QDs as a label for biorecognition events and using the photocurrent generated in the presence of auxiliary electron donor/ acceptor substances as a readout signal. The second approach uses the biorecognition event as an active component that activates the formation of the photocurrent. The use of CdS QDs as photoelectrochemical labels is exemplified in Figure 7B with the development of a cocaine aptasensor.57 The two anticocaine aptamer subunits were used to assemble the sensor. One aptamer subunit was immobilized on a Au electrode, whereas the second subunit was functionalized with the CdS QD. In the presence of cocaine, the respective aptamersubstrate complex was formed on the electrode, and the CdS QDs acted as labels for the generation of the photocurrent in the presence of the coadded electron donor, triethanol amine. That is, the ejection of the conduction band electrons into the electrode was accompanied by the reduction of the valenceband holes by electrons donated by the electron donor, giving rise to the generation of the photocurrent. As the coverage of the cocaine aptamer complex on the electrode increases upon elevating the concentration of the cocaine analyte, the resulting photocurrent intensity provides a quantitative measure for the concentration of the analyte (Figure 7C). The use of CdS QDs as labels for biocatalytic transformations was also demonstrated with the development of a photoelectrochemical sensor for tyrosinase58 (Figure 7D). CdS QDs were functionalized with a capping layer consisting of tyrosine methyl ester. Tyrosinasecatalyzed oxidation of the tyrosine units to methyl ester L-DOPA residues yielded ligand-capped QDs that associated with a boronic acid-functionalized Au electrode. The resulting CdS QD-functionalized electrode generated an anodic photocurrent that reflected the activity of tyrosinase. Semiconductor-functionalized electrodes were also conjugated to enzymes, resulting in the formation of photocurrents as a result of biocatalytic processes. A CdS-based photoelectrochemical sensor for the analysis of the activity of acetylcholine esterase, AChE, and the detection of inhibitors of the enzyme (nerve gas analogues) was developed.59 The enzyme AChE was covalently linked to a CdS QDs-modified electrode (Figure 7E). The biocatalyzed hydrolysis of acetylthiocholine (6) yielded thiocholine (7), which acted as a hole scavenger of the exciton generated in the QDs. Thus, the resulting photocurrent related directly to the concentration of the acetylthiocholine substrate 6 (Figure 7F). In the presence of the AChE inhibitor, 1,5-bis (4-allyldimethylammonium phenyl) pentane-3-one (8), the formation of the photocurrent was blocked, demonstrating the ability of the system to probe AChE inhibitors (Figure 7G). Rinsing off of the inhibitor restored the photoelectrochemical activity of the enzyme-modified electrode, revealing the recycling of the sensor. Perspectives. Substantial progress in the application of semiconductor QDs for optical or photoelectrochemical biosensing was achieved in the past few years. The unique luminescence features of semiconductor QDs were vastly used for labeling and imaging of biorecognition events. By combining photophysical mechanisms, such as FRET, ET, and CRET, with the properties and functions of QDs, diverse biosensing platforms were developed. The photostability of semiconductor QDs, as compared to conventional dyes, and the feasibility to load QDs with a high
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content of molecular-active components are certainly major advantages for the optical analysis of biological events, particularly, under harsh, long-term, characterization of intracellular processes.
Semiconductor QDs act as photoelectrochemical labels for biosensing. The future perspectives of bioanalytical applications of QDs rest, however, in the development of novel multiplexed analysis schemes, and particularly, in the application of functional biomoleculeQDs hybrids for probing intracellular transformations and imaging target domains in cells and tissues. Specifically, one could use the photoinduced electron trasfer functions of different semiconductor QDs, e.g., TiO2 or CdS, to yield reactive oxygen species in cells, and to probe the effect of antioxidants on the reactive species, using appropriately functionalized sensing QDs. These efforts will require an increase in the arsenal of functionalized QDs to probe other intracellular targets. Also, the development of new methodologies to deliver and incorporate the QDs into biological media and to initiate a scientific effort that transfers innovative fundamental science from in vitro experiments to in vivo environments is essential. The rapid discovery of new molecular or macromolecular biomarkers for various diseases and the identification of new biomolecular receptors or man-made nucleic acid (aptamer) receptors provide, however, new opportunities for the intracellular application of QDs for future nanomedicine. Furthermore, the chemical modification of QDs enables not only their use as optical materials for bioanalytical sensing applications, but one may envision their cooperative function as carriers for chemical components. Such hybrid sensor-carrier nanocomposites may find further applications as autonomous “sense and treat” systems.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Tel: +972-2-6585272. Fax: +972-2-6527715.
’ BIOGRAPHIES Ronit Freeman is a Ph.D. student in the laboratory of Professor I. Willner. Her research involves biocatalytical applications of quantum dots, DNA nanostructures, and the application of nanoparticles for probing intracellular processes. She holds the Clore Foundation Scholars Award for Ph.D. studies. Dr. Bilha Willner completed her Ph.D. studies at the Hebrew University of Jerusalem in 1982 in the area of organometallic chemistry. She has acted as a Senior Research Associate in the laboratory of Prof. I. Willner since 1986. Her scientific interests include bioelectrochemistry, biosensors, assembly of nanoparticles on surfaces and nanobiotechnology. Itamar Willner is a Professor of Chemistry at the Hebrew University of Jerusalem. His research interests include bioelectronics, nanobiotechnology, molecular and biomolecular machines. He has coauthored over 600 papers and monographs. He received the Israel Prize in Chemistry (2002), the Rothschild 2675
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The Journal of Physical Chemistry Letters Prize (2008), and the EMET Prize (2008). He is a member of the Israel Academy of Sciences and the German National Academy German National Academy of Sciences Leopoldina.
’ ACKNOWLEDGMENT Parts of our research of biomoleculequantum dot hybrid systems for biocatalytic applications are supported by The Israel Science Foundation and by The Israel Scientific research Ministry of Science and Technology (TaiwanIsrael scientific research cooperation programme). ’ REFERENCES (1) Alivisatos, A. P. Semiconductor Clusters, Nanocrystals, and Quantum Dots. Science 1996, 271, 933–937. (2) Nirmal, M.; Brus, L. Luminescence Photophysics in Semiconductor Nanocrystals. Acc. Chem. Res. 1999, 32, 407–414. (3) Wu, X. Y.; Liu, H. J.; Liu, J. Q.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N. F.; Peale, F.; Bruchez, M. P. Immunofluorescent Labeling of Cancer Marker Her2 and Other Cellular Targets with Semiconductor Quantum Dots. Nat. Biotechnol. 2003, 21, 41–46. (4) Alivisatos, P. The Use of Nanocrystals in Biological Detection. Nat. Biotechnol. 2004, 22, 47–52. (5) Gill, R.; Zayats, M.; Willner, I. Semiconductor Quantum Dots for Bioanalysis. Angew. Chem., Int. Ed. 2008, 47, 7602–7625. (6) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Quantum Dot Bioconjugates for Imaging, Labelling and Sensing. Nat. Mater. 2005, 4, 435–446. (7) Peng, Z. A.; Peng, X. Z. Formation of High-Quality CdTe, CdSe, and CdS Nanocrystals Using CdO as Precursor. J. Am. Chem. Soc. 2001, 123, 183–184. (8) Yu, W. W.; Chang, E.; Falkner, J. C.; Zhang, J. Y.; Al-Somali, A. M.; Sayes, C. M.; Johns, J.; Drezek, R.; Colvin, V. L. Forming Biocompatible and Nonaggregated Nanocrystals in Water Using Amphiphilic Polymers. J. Am. Chem. Soc. 2007, 129, 2871–2879. (9) Lianos, P.; Thomas, J. K. Cadmium Sulfide of Small Dimensions Produced in Inverted Micelles. Chem. Phys. Lett. 1986, 125, 299–302. (10) Murray, C. B.; Norris, D. J.; Bawendi, M. G. Synthesis and Characterization of Nearly Monodisperse CdE (E = S, Se, Te) Semiconductor Nanocrystallites. J. Am. Chem. Soc. 1993, 115, 8706–8715. (11) Willard, D. M.; Carillo, L. L.; Jung, J.; Van Orden, A. CdSeZnS Quantum Dots as Resonance Energy Transfer Donors in a Model ProteinProtein Binding Assay. Nano Lett. 2001, 1, 469–474. (12) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F. V.; Bawendi, M. G. Self-Assembly of CdSeZnS Quantum Dot Bioconjugates Using an Engineered Recombinant Protein. J. Am. Chem. Soc. 2000, 122, 12142–12150. (13) Liu, W.; Howarth, M.; Greytak, A. B.; Zheng, Y.; Nocera, D. G.; Ting, A. Y.; Bawendi, M. G. Compact Biocompatible Quantum Dots Functionalized for Cellular Imaging. J. Am. Chem. Soc. 2008, 130, 1274– 1284. (14) B€aumle, M.; Stamou, D.; Segura, J. M.; Hovius, R.; Vogel, H. Highly Fluorescent Streptavidin-Coated CdSe Nanoparticles: Preparation in Water, Characterization, and Micropatterning. Langmuir 2004, 20, 3828–3831. (15) Medintz, I. L.; Clapp, A. R.; Brunel, F. M.; Tiefenbrunn, T.; Uyeda, H. T.; Chang, E. L.; Deschamps, J. R.; Dawson, P. E.; Mattoussi, H. Proteolytic Activity Monitored by Fluorescence Resonance Energy Transfer through Quantum-DotPeptide Conjugates. Nat. Mater. 2006, 5, 581–589. (16) Goldman, E. R.; Clapp, A. R.; Anderson, G. P.; Uyeda, H. T.; Mauro, J. M.; Medintz, I. L.; Mattoussi, H. Multiplexed Toxin Analysis Using Four Colors of Quantum Dot Fluororeagents. Anal. Chem. 2004, 76, 684–688. (17) Gerion, D.; Chen, F. Q.; Kannan, B.; Fu, A. H.; Parak, W. J.; Chen, D. J.; Majumdar, A.; Alivisatos, A. P. Room-Temperature Single-
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Nucleotide Polymorphism and Multiallele DNA Detection Using Fluorescent Nanocrystals and Microarrays. Anal. Chem. 2003, 75, 4766– 4772. (18) Kim, J. H.; Chaudhary, S.; Ozkan, M. Multicolour Hybrid Nanoprobes of Molecular Beacon Conjugated Quantum Dots: FRET and Gel Electrophoresis-Assisted Target DNA Detection. Nanotechnology 2007, 18, 195105–195111. (19) Tuerk, C.; Gold, L. Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase. Science 1990, 249, 505–510. (20) Ellington, A. D.; Szostak, J. W. In vitro Selection of RNA Molecules that Bind Specific Ligands. Nature 1990, 346, 818–822. (21) Willner, I.; Zayats, M. Electronic Aptamer-Based Sensors. Angew. Chem., Int. Ed. 2007, 46, 6408–6418. (22) Freeman, R.; Li, Y.; Tel-Vered, R.; Sharon, E.; Elbaz, J.; Willner, I. Self-Assembly of Supramolecular Aptamer Structures for Optical or Electrochemical Sensing. Analyst 2009, 134, 653–656. (23) Travascio, P.; Witting, P. K.; Mauk, A. G.; Sen, D. The Peroxidase Activity of a HeminDNA Oligonucleotide Complex: Free Radical Damage to Specific Guanine Bases of the DNA. J. Am. Chem. Soc. 2001, 123, 1337–1348. (24) Weizmann, Y.; Beissenhirtz, M. K.; Cheglakov, Z.; Nowarski, R.; Kotler, M.; Willner, I. A Virus Spotlighted by an Autonomous DNA Machine. Angew. Chem., Int. Ed. 2006, 45, 7384–7388. (25) Xiao, Y.; Pavlov, V.; Niazov, T.; Dishon, A.; Kotler, M.; Willner, I. Catalytic Beacons for the Detection of DNA and Telomerase Activity. J. Am. Chem. Soc. 2004, 126, 7430–7431. (26) Li, D.; Shlyahovsky, B.; Elbaz, J.; Willner, I. Amplified Analysis of Low Molecular Weight Substrates or Proteins by the Self-Assembly of DNAzymeAptamer Conjugates. J. Am. Chem. Soc. 2007, 129, 5804– 5805. (27) Sharon, E.; Freeman, R.; Willner, I. CdSe/ZnS Quantum DotsG-Quadruplex/Hemin Hybrids as Optical DNA Sensors and Aptasensors. Anal. Chem. 2010, 82, 7073–7077. (28) Pavlov, V.; Xiao, Y.; Gill, R.; Dishon, A.; Kotler, M.; Willner, I. Amplified Chemiluminescence Surface Detection of DNA and Telomerase Activity Using Catalytic Nucleic Acid Labels. Anal. Chem. 2004, 76, 2152–2156. (29) Freeman, R.; Liu, X.; Willner, I. Chemiluminescent and Chemiluminescence Resonance Energy Transfer (CRET) Detection of DNA, Metal Ions and Aptamer-Substrate Complexes Using Hemin/ G-Quadruplexes and CdSe/ZnS Quantum Dots. J. Am. Chem. Soc. 2011, 133, 11597–11604. (30) Shi, L. F.; De Paoli, V.; Rosenzweig, N.; Rosenzweig, Z. Synthesis and Application of Quantum Dots FRET-Based Protease Sensors. J. Am. Chem. Soc. 2006, 128, 10378–10379. (31) Medintz, Z. L.; Clapp, A. R.; Brunel, F. M.; Tiefenbrunn, T.; Uyeda, H. T.; Chang, E. L.; Deschamps, J. R.; Dawson, P. E.; Mattoussi, H. Proteolytic Activity Monitored by Fluorescence Resonance Energy Transfer through Quantum-DotPeptide Conjugates. Nat. Mater. 2006, 5, 581–589. (32) Freeman, R.; Finder, T.; Gill, R.; Willner, I. Probing Protein Kinase (CK2) and Alkaline Phosphatase with CdSe/ZnS Quantum Dots. Nano Lett. 2010, 10, 2192–2196. (33) Ghadiali, J. E.; Cohen, B. E.; Stevens, M. M. Protein KinaseActuated Resonance Energy Transfer in Quantum DotPeptide Conjugates. ACS Nano 2010, 4, 4915–4919. (34) Ghadiali, J. E.; Lowe, S. B.; Stevens, M. M. Quantum-Dot-Based FRET Detection of Histone Acetyltransferase Activity. Angew. Chem., Int. Ed. 2011, 50, 3417–3420. (35) Patolsky, F.; Gill, R.; Weizmann, Y.; Mokari, T.; Banin, U.; Willner, I. Lighting Up the Dynamics of Telomerization and DNA Replication by CdSeZnS Quantum Dots. J. Am. Chem. Soc. 2003, 125, 13918–13919. (36) Gill, R.; Willner, I.; Shweky, I.; Banin, U. Fluorescence Resonance Energy Transfer in CdSe/ZnSDNA Conjugates: Probing Hybridization and DNA Cleavage. J. Phys. Chem. B 2005, 109, 23715–23719. (37) Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West, M. D.; Ho, P. L. C.; Coviello, G. M.; Wright, W. E.; Weinrich, S. L.; Shay, 2676
dx.doi.org/10.1021/jz2006572 |J. Phys. Chem. Lett. 2011, 2, 2667–2677
The Journal of Physical Chemistry Letters J. W. Specific Association of Human Telomerase Activity with Immortal Cells and Cancer. Science 1994, 266, 2011–2015. (38) Shay, J. W.; Bacchetti, S. A Survey of Telomerase Activity in Human Cancer. Eur. J. Cancer 1997, 33, 787–791. (39) Angeletti, C.; Khomitch, V.; Halaban, R.; Rimm, D. L. Novel Tyramide-Based Tyrosinase Assay for the Detection of Melanoma Cells in Cytological Preparations. Diagnos. Cytopathol. 2004, 31, 33–37. (40) Gill, R.; Freeman, R.; Xu, J. P.; Willner, I.; Winograd, S.; Shweky, I.; Banin, U. Probing Biocatalytic Transformations with CdSe-ZnS QDs. J. Am. Chem. Soc. 2006, 128, 15376–15377. (41) Parak, W. J.; Boudreau, R.; Le Gros, M.; Gerion, D.; Zanchet, D.; Micheel, C. M.; Williams, S. C.; Alivisatos, A. P.; Larabell, C. Cell Motility and Metastatic Potential Studies Based on Quantum Dot Imaging of Phagokinetic Tracks. Adv. Mater. 2002, 14, 882–885. (42) Smith, B. R.; Cheng, Z.; De, A.; Koh, A. L.; Sinclair, R.; Gambhir, S. S. Real-Time Intravital Imaging of RGDQuantum Dot Binding to Luminal Endothelium in Mouse Tumor Neovasculature. Nano Lett. 2008, 8, 2599–2606. (43) Silver, J.; Ou, W. Photoactivation of Quantum Dot Fluorescence Following Endocytosis. Nano Lett. 2005, 5, 1445–1449. (44) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Intracellular Delivery of Quantum Dots for Live Cell Labeling and Organelle Tracking. Adv. Mater. 2004, 16, 961–966. (45) Chen, F. Q.; Gerion, D. Fluorescent CdSe/ZnS Nanocrystal Peptide Conjugates for Long-term, Nontoxic Imaging and Nuclear Targeting in Living Cells. Nano Lett. 2004, 4, 1827–1832. (46) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. In Vivo Imaging of Quantum Dots Encapsulated in Phospholipid Micelles. Science 2002, 298, 1759–1762. (47) Ji, X. J.; Zheng, J. Y.; Xu, J. M.; Rastogi, V. K.; Cheng, T. C.; DeFrank, J. J.; Leblanc, R. M. (CdSe)ZnS Quantum Dots and Organophosphorus Hydrolase Bioconjugate as Biosensors for Detection of Paraoxon. J. Phys. Chem. B 2005, 109, 3793–3799. (48) Bagalkot, V.; Zhang, L.; Levy-Nissenbaum, E.; Jon, S.; Kantoff, P. W.; Langer, R.; Farokhzad, O. C. Quantum DotAptamer Conjugates for Synchronous Cancer Imaging, Therapy, and Sensing of Drug Delivery Based on Bi-Fluorescence Resonance Energy Transfer. Nano Lett. 2007, 7, 3065–3070. (49) Chen, A. A.; Derfus, A. M.; Khetani, S. R.; Bhatia, S. N. Quantum Dots to Monitor RNAi Delivery and Improve Gene Silencing. Nucleic Acids Res. 2005, 33, e190. (50) Freeman, R.; Gill, R.; Shweky, I.; Kotler, M.; Banin, U.; Willner, I. Biosensing and Probing of Intracellular Metabolic Pathways by NADH-Sensitive Quantum Dots. Angew. Chem., Int. Ed. 2009, 48, 309–313. (51) McLaurin, E. J.; Greytak, A. B.; Bawendi, M. G.; Nocera, D. G. Two-Photon Absorbing Nanocrystal Sensors for Ratiometric Detection of Oxygen. J. Am. Chem. Soc. 2009, 131, 12994–13001. (52) Wong, C.; Stylianopoulos, T.; Cui, J.; Martin, J.; Chauhan, V. P.; Jiang, W.; Popovic, Z.; Jain, R. K.; Bawendi, M. G.; Fukumura, D. Multistage Nanoparticle Delivery System for Deep Penetration into Tumor Tissue. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 2426–2431. (53) Medintz, I. L.; Stewart, M. H.; Trammell, S. A.; Susumu, K.; Delehanty, J. B.; Mei, B. C.; Melinger, J. S.; Blanco-Canosa, J. B.; Dawson, P. E.; Mattoussi, H. Quantum-Dot/Dopamine Bioconjugates Function As Redox Coupled Assemblies for in Vitro and Intracellular pH Sensing. Nat. Mater. 2010, 9, 676–684. (54) Clarke, S. J.; Hollmann, C. A.; Zhang, Z.; Suffern, D.; Bradforth, S. E.; Dimitrijevic, N. M.; Minarik, W. G.; Nadeau, J. L. Photophysics of Dopamine-Modified Quantum Dots and Effects on Biological Systems. Nat. Mater. 2006, 5, 409–417. (55) Kamat, P. V. Meeting the Clean Energy Demand: Nanostructure Architectures for Solar Energy Conversion. J. Phys. Chem. C 2007, 111, 2834–2860. (56) Gr€atzel, M. Recent Advances in Sensitized Mesoscopic Solar Cells. Acc. Chem. Res. 2009, 42, 1788–1798. (57) Golub, E.; Pelossof, G.; Freeman, R.; H. Zhang, H.; Willner, I. Electrochemical, Photoelectrochemical and Surface Plasmon Resonance
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Detection of Cocaine Using Supramolecular Aptamer Complexes and Metallic or Semiconductor Nanoparticles. Anal. Chem. 2009, 81, 9291– 9298. (58) Yildiz, H. B.; Freeman, R.; Gill, R.; Willner, I. Electrochemical, Photoelectrochemical and Piezoelectric Analysis of Tyrosinase Activity by Functionalized Nanoparticles. Anal. Chem. 2008, 80, 2811–2816. (59) Pardo-Yissar, V.; Katz, E.; Wasserman, J.; Willner, I. Acetylcholine Esterase-Labeled CdS Nanoparticles on Electrodes: Photoelectrochemical Sensing of the Enzyme Inhibitors. J. Am. Chem. Soc. 2003, 125, 622–623.
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