Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A

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Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications Niko Hildebrandt,§,○ Christopher M. Spillmann,†,○ W. Russ Algar,∥,○ Thomas Pons,⊥ Michael H. Stewart,‡ Eunkeu Oh,‡,# Kimihiro Susumu,‡,# Sebastian A. Díaz,†,∇ James B. Delehanty,† and Igor L. Medintz*,† †

Center for Bio/Molecular Science and Engineering, Code 6900, and ‡Optical Sciences Division, Code 5600, U.S. Naval Research Laboratory, Washington, DC 20375, United States § NanoBioPhotonics Institut d’Electronique Fondamentale (I2BC), Université Paris-Saclay, Université Paris-Sud, CNRS, 91400 Orsay, France ∥ Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada ⊥ LPEM; ESPCI Paris, PSL Research University; CNRS; Sorbonne Universités, UPMC, F-75005 Paris, France # Sotera Defense Solutions, Inc., Columbia, Maryland 21046, United States ∇ American Society for Engineering Education, Washington, DC 20036, United States ABSTRACT: Luminescent semiconductor quantum dots (QDs) are one of the more popular nanomaterials currently utilized within biological applications. However, what is not widely appreciated is their growing role as versatile energy transfer (ET) donors and acceptors within a similar biological context. The progress made on integrating QDs and ET in biological configurations and applications is reviewed in detail here. The goal is to provide the reader with (1) an appreciation for what QDs are capable of in this context, (2) how this field has grown over a relatively short time span, and, in particular, (3) how QDs are steadily revolutionizing the development of new biosensors along with a myriad of other photonically active nanomaterial-based bioconjugates. An initial discussion of QD materials along with key concepts surrounding their preparation and bioconjugation is provided given the defining role these aspects play in the QDs ability to succeed in subsequent ET applications. The discussion is then divided around the specific roles that QDs provide as either Förster resonance energy transfer (FRET) or charge/electron transfer donor and/or acceptor. For each QD-ET mechanism, a working explanation of the appropriate background theory and formalism is articulated before examining their biosensing and related ET utility. Other configurations such as incorporation of QDs into multistep ET processes or use of initial chemical and bioluminescent excitation are treated similarly. ET processes that are still not fully understood such as QD interactions with gold and other metal nanoparticles along with carbon allotropes are also covered. Given their maturity, some specific applications ranging from in vitro sensing assays to cellular imaging are separated and discussed in more detail. Finally a perspective on how this field will continue to evolve is provided.

CONTENTS 1. Introduction 2. Brief History 3. Quantum Dots, Surface Functionalization, Bioconjugation, and Relevant Considerations 3.1. Quantum Dot Materials 3.2. Surface Functionalization 3.2.1. Surface Functionalization Methods 3.2.2. PEG versus Zwitterionic Solubilizing Ligands 3.2.3. Direct Aqueous and Biological Synthesis 3.3. Bioconjugation 3.4. Definitions

© 2016 American Chemical Society

4. Overview of Energy Transfer Mechanisms and Signal Transduction 4.1. Energy Transfer Mechanisms 4.2. Signal Transduction 4.3. Pairing of Donor/Acceptor Materials 5. Fö rster Resonance Energy Transfer 5.1. Theory and Formalism 5.2. Quantum Dots as Donor 5.2.1. Small Molecule Sensors

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Special Issue: Light Harvesting Received: January 13, 2016 Published: June 30, 2016 536

DOI: 10.1021/acs.chemrev.6b00030 Chem. Rev. 2017, 117, 536−711

Chemical Reviews 5.2.2. Immunosensors 5.2.3. Enzymatic Sensors 5.2.4. Nucleic Acid−Based Sensors 5.2.5. Multistep Energy Transfer Configurations 5.2.6. Heterogeneous Analysis 5.2.7. Other Sensors and Bioconjugates 5.3. Quantum Dots as Acceptor 5.3.1. Use of Lanthanide Complex Donors and Time-Resolved Formats 5.3.2. Sensors and Assays for Diagnostics and Imaging 5.3.3. Quantum Dots as Relays 5.3.4. Spectroscopic Rulers 5.3.5. BRET/CRET 5.3.6. Upconversion Nanoparticles 5.4. Quantum Dot to Quantum Dot FRET 6. Energy Transfer with Gold and Other Metals 6.1. Theory and Formalism 6.2. Functional Bioconjugates and Sensors 6.2.1. Initial Bioconjugates 6.2.2. Sensors 7. Energy Transfer with Carbon Allotropes 7.1. Sensors 7.2. Other Bioconjugates 8. Electron Transfer 8.1. Theory and Formalism 8.2. Quantum Dots as Donor 8.3. Quantum Dots as Acceptor 8.4. Special Case of Benzenediols 9. Single Molecule Quantum Dot Energy Transfer Studies 10. Cellular-Based Sensing 10.1. Cytosolic Targets and Intracellular Molecular Assembly 10.1.1. Ions/Cofactors 10.1.2. pH 10.1.3. Intracellular Assembly and Disassembly 10.2. Plasma Membrane and Related Extracellular Processes 10.2.1. Membrane Dynamics 10.2.2. Receptor−Ligand Interaction and Endocytosis 10.2.3. Activity of Secreted Proteases 11. Photodynamic Therapy 12. Applications with Diagnostic and Sensing Devices 12.1. Microfluidics 12.2. Smartphones and Custom Bioanalytical Devices 13. Light Harvesting 14. Related and Miscellaneous Structures/Processes 14.1. Electrode Systems Incorporating Quantum Dots 14.2. Relaying Electrons to Enzymes and Cofactors 14.3. Hydrogen Production 15. Conclusions and Outlook Author Information Corresponding Author Author Contributions Notes

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Biographies Acknowledgments List of Abbreviations References Note Added in Proof

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1. INTRODUCTION The most basic definition of nanotechnology is often given as the manipulation of matter on the atomic, molecular, and supramolecular scales. A slightly more focused definition with potential legal and regulatory ramifications has been established by the National Nanotechnology Initiative, which suggests that nanotechnology is “the manipulation of matter with at least one dimension sized from 1 to 100 nm.”1 Along with these definitions, we subscribe to a more colloquial working definition of nanotechnology, which is technology where biology, chemistry, and/or physics converge at the nanoscale to create new “value-added” materials that are capable of far more than the corresponding bulk materials or constituent molecules individually. This definition is also more closely related to how the public perceives nanotechnology, whether from a beneficial or detrimental perspective.2 Examples of the fruition of such nanotechnologies could include, as an example within the context of oncology, nanoscale biological-nanomaterial (NM) theranostic devices capable of simultaneously providing in vivo tumor targeting, localized drug delivery, and tumor marker biosensing, all while also providing contrast during external imaging.3−5 Colloidal semiconductor nanocrystals, which are commonly referred to as quantum dots (QDs), are one of the most popular, prolific, and promising NMs, particularly from the standpoint of impacting biology. Value can be added to the inherent properties of QDs by modifying these materials with biological moieties and coupling their properties to those of other molecules and NMs through both bioconjugation and energy transfer processes. With these definitions and context in mind, we review in depth the role luminescent QDs have played in energy transfer (ET), within the specific context of QD-based bioconjugates and their biological applications. Although QDs are clearly at the intersection of several scientific disciplines, limiting this review to biological contexts helps narrow its scope and separate it from the vast role that the combination of QDs and ET are playing within solar power and energy harvesting, electronics, photonics, and several other related fields.6−12 Almost every fluorescent nanoparticle (NP) material, including those synthesized from silicon, gold, silver, dyeimpregnated nanospheres, rare-earth upconversion NPs, carbon allotropes such as carbon dots and graphene dots, organic polymers, and many others materials, have all been correctly or incorrectly referred to as a “quantum dot” at one time or another and perhaps have already been demonstrated within an ET application. We limit this review to what some consider the “original” QD material, namely those prepared with core, core− shell, or core−multishell configurations from binary, tertiary, alloyed, or other semiconductors in nanocrystalline form; see Figure 1. In addition we limit the focus here to II−VI (e.g., CdTe and CdSe), III−V (e.g., InP), and related semiconductors in colloidal form, as opposed to epitaxial QDs. We note that silicon and germanium (group IV) nanocrystals also display interesting quantum confinement effects and some size-dependent emission properties, but these are not as well controlled synthetically as the previous.13,14 Moreover, as their

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approach values 10−100× greater than that of dyes; and some of the largest two-photon action (TPA) cross sections available.15−26 Cumulatively, these properties make QDs quite superior to conventional fluorophores within challenging formats such as in vivo imaging and multiplexing. Moreover, as specifically highlighted in Figure 2, semiconductor QDs are also currently the most unique and versatile materials available for engaging in both Förster resonance energy transfer (FRET) and electron/charge transfer (eT and CT, respectively). QDs have already been demonstrated to be excellent FRET and ET donors for a wide array of structurally and photophysically diverse organic dyes, fluorescent polymers, fluorescent proteins, and noble metal NPs.27−33 QDs have also been shown to be excellent FRET acceptors for long-lifetime rare earth metal chelates, other QDs, fluorescent polymers, and the products of bioluminescence-generating enzymatic reactions and chemiluminescence-generating reactions (Figure 2A).30,33−38 Due to their inherent electronic properties, and depending upon the electroactive species with which they are combined, QDs can also function as quite effective CT donors and acceptors (Figure 2B).39 Although different organic dye families can engage in all these types of activities to some extent as well,40 no one single organic dye, molecular fluorophore, or fluorescent protein can do all as effectively and efficiently while it is quite probable that a single QD sample could. Despite requiring significant preparatory efforts for use in biological and aqueous environments (discussed below), QDs also present several useful properties in the form of a bioconjugate. Their non-trivial size and surface area (even at the nanoscale) means that QDs can typically be bioconjugated with multiple copies of a peptide, nucleic acid, drug, protein, or other biological either individually or in combination. When this conjugation is properly implemented, control can be exerted over the number and orientation of such species displayed on the QD.41,42 This multivalency can potentially translate into higher chemical stability, avidity, and sensitivity in a biological assay. Moreover, such composite biological assemblies also provide the unique ability to create and access discrete QD configurations where both the ratio of a given donor or acceptor and its separation distance can be incrementally changed relative to the QD. The same set of properties is not easily achieved with layer-by-layer (LBL) deposition, spin coating, or other chemical growth techniques. All of these properties in aggregate suggest that QDs are uniquely positioned to be not only a very powerful component, but perhaps even a uniquely enabling NM for ET-based biosensors and similarly functional composites. The versatility of QDs has indeed been proven over the past decade or so with ever more reports appearing each year. A search of Google Scholar with the keywords “quantum dots AND biosensors” or “quantum dots AND energy transfer AND biosensors” returns >35,000 and >25,000 results, respectively. Interestingly, searching “quantum dots AND energy transfer” returns >600,000 results reflecting the roles that ET with these materials have in other processes of interest.43 A strong case can also be made that QDs are an invaluable prototypical NM for learning important lessons about how to interface NPs in general with biological molecules and/or environments, an example being the contributions of QDs to the burgeoning field of NP-mediated drug delivery.44−47 Bright QD PL is easy to follow in vitro and in vivo, and the maturity and advanced development of QD synthetic and surface chemistries make them valuable tools for developing new NP solubilization and

Figure 1. (A) Cartoon and TEM image of a CdSe/ZnS QD. (B) Cartoon, photograph, and PL emission spectra illustrating progressive color changes of CdSe/ZnS with increasing nanocrystal size. (C) Qualitative changes in QD energy levels with increasing nanocrystal size. Band gap energies, Eg, were estimated from the PL spectra. Conduction (CB) and valence (VB) bands of bulk CdSe are shown for comparison. The energy scale is expanded as 10E for clarity. Reproduced from ref 17. Copyright 2011 American Chemical Society.

utilization for energy transfer in a biological context is extremely limited, we do not consider them in the current discussion. The intent of all of the foregoing exclusions is again to help focus and limit the content of this review so as to allow it to be more comprehensive and detailed. So why focus on the intersection of QDs and ET in the context of biological applications or QD-based bioconjugates? From the perspective of fluorescent labeling, QDs have many desirable properties, including their unrivaled size- and/or material dependent, spectrally narrow, and symmetrical photoluminescence (PL) emission profiles (see Figure 1); high quantum yields (QYs); unrivaled photo- and chemical stability; broad absorption cross sections that increase steadily toward the UV from their first absorption band, and which can 538


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Figure 2. QDs can engage in FRET and CT in the role of either a donor or acceptor depending upon configuration. (A) QDs are good FRET donors for fluorescent protein (FP), dye, and gold nanoparticle (AuNP) acceptors. The dashed circle represents an arbitrary Förster distance (R0) measured from the QD center. The scale at the right indicates how the effective R0 (i.e., EFRET = 0.5) proportionally increases as the number of proximal acceptors (a) increases. Conversely, QDs can function as acceptors for long-lifetime terbium (Tb) complexes and bioluminescent luciferase enzyme (Luc) donors. (B) CT quenching is an alternative method of modulating QD PL: (i) an electron acceptor (e.g., quinone) has an unoccupied energy level intermediate in energy to the 1Sh and 1Se band-edge states to which the excited QD transfers an electron, (ii) an electron donor (e.g., ruthenium phenanthroline-Ru2+) has an occupied intermediate energy level and transfers an electron to the QD. Charge transfer inhibits radiative recombination of the exciton. Both redox active species are illustrated as peptide conjugates that could be assembled to the surface of QDs. Note: processes (i) and (ii) can occur independently when the QD is coupled to the requisite electron donor or acceptor. The image is not meant to imply that they occur in parallel. Reproduced from ref 17. Copyright 2011 American Chemical Society.

bioconjugation techniques.41,48,49 There is also growing interest in identifying and understanding newly emergent or previously uncharacterized processes that only occur at the bio-NP interface. These processes span a broad spectrum, from those that generally appear to be detrimental, such as protein corona formation,50−53 to those that may be beneficial and exploitable, such as localized increases in enzymatic catalytic rates.54 The ability to utilize QDs and ET to understand all of these processes and improve NMs in general will certainly be beneficial in the long term. Thus, we come full circle and return to the definition of nanotechnology as the intersection of biology, chemistry, materials, and/or physics at the nanoscale to create new “valueadded” materials. The following sections review the progress made on integrating QDs and ET in many of its photophysical manifestations in the context of biological configurations and applications. There are a plethora of other QD-based biological materials described in the literature; however, our discussion focuses only on those materials falling under the unifying theme of ET with QDs. Our goal is to provide the reader with an appreciation for how this field has grown in a relatively short time, the capabilities of QDs in this context, and, in particular, how QDs are steadily revolutionizing the development of new biosensors along with a myriad of other photonically active NM bioconjugates. We utilize figures and images from much of the reviewed literature to richly illustrate the concepts discussed. The review begins with a brief discussion of QD materials including their preparation and bioconjugation, as these modifications play such a large and defining role in subsequent ET applications. Key concepts and issues are presented to allow the reader to appreciate how interfacial chemistry influences both an intended application and the success of that application. As delineated in Figure 2, we then divide the discussion according to the specific role of the QD in a given ET application, including that of FRET donor or acceptor and

CT donor or acceptor. For each QD-ET mechanism, a working explanation of the background theory and formalism is provided with a critical discussion of their biosensing and related ET utility. We also include ET processes that are still not fully understood or defined, such as QD interactions with gold and other metal NPs or carbon allotropes. As warranted by the maturity and scope of this field, the review is further organized into specific domains of applications, ranging from in vitro sensing assays to cellular imaging. Other configurations, such as incorporation of QDs into multistep ET processes or use of initial chemical and bioluminescent excitation, are also treated in their own sections. Due to overlap in subject matter, we note that some examples from the literature are discussed in more than one section so as to highlight different aspects. For example, a particular QD-ET sensor may be discussed in one section under QD donors in the context of its biological target, and its application within cells discussed in another section. This duplication allows each section to cover its intended focus and stand alone. Given the breadth of literature available, we focus on many of the primary examples and realize that we cannot cover every possible publication. Omission from this review is not meant to detract from the importance of any one contribution, and we extend our apologies for any and all omissions.

2. BRIEF HISTORY It is quite probable that, following the initial syntheses and subsequent photophysical characterization of colloidal QDs, some of those ensemble materials did indeed engage in resonance energy transfer (RET, and most likely homoFRET in this scenario) while being investigated in layered and similarly structured assemblies.55−57 However, the focus during those early years was to understand the fundamental properties of QDs, which, when coupled with the short viable experimental lifetimes and immature properties of initial QD materials, left 539


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RET unexplored. The first in-depth exploration of RET between QD donors and QD acceptors is described in two papers originating from the Bawendi Lab in 1996.58,59 Photophysical interrogation of close-packed CdSe QD solids prepared from a mixture of small and large nanocrystals showed all the hallmarks of classical FRET including a quenching of the PL lifetime of the smaller QDs (donors) that was accompanied by a concomitant increase in the PL lifetime of the larger QDs (acceptors). The long-range RET observed here was analyzed directly within the context of Förster theory assuming an underpinning of QD-QD dipole coupling. Just two years later, the ground-breaking reports of QD preparations that were both stable in aqueous media and useful as cellular probes began the next stage of development.60,61 One new goal during this phase was centered on utilizing biological molecules and their interactions to create more “controlled” QD FRET configurations to engage in some form of biosensing. Control in this context meant that the separation distance between the QD and a FRET acceptor could be predetermined by the size of the biomolecule(s) utilized, and biosensing would seek to exploit the drastic change in FRET efficiency expected when the QD and this acceptor went from close proximity to partial or complete separation. One of the first important steps in this development process was reported by Van Orden’s group in 2001 using QD donors and a discrete dye acceptor within the context of a biotin (Bt)-avidin binding interaction.62 Importantly, they suggested the ability to sizetune QD emission to optimize FRET with the acceptor as being a unique and important component for these types of composite materials. Another important step was reported by the Studer group in 2002.63 They coupled green CdTe QDs (donor) to an antibody and red QDs (acceptor) to the antigen and showed that the immunocomplex resulted in FRET with all the expected photophysical manifestations. Around the same time, Mattoussi and co-workers at the U.S. Naval Research Laboratory showed that dark quencher-labeled recombinantly modified proteins could electrostatically attach to QDs and controllably modulate or quench the QD donor PL in a manner that directly reflected the number of protein-quenchers attached on average to the QDs.64 This process was shown to function in both solution-phase and solid-phase configurations. This paper was followed about a year later by three sequential papers that some consider the initial defining demonstrations of QD-based FRET within biological configurations. In 2003, a displacement-based QD FRET sensor for quantitative detection of maltose was reported by Mattoussi and colleagues at the Naval Research Lab. Here, QDs were ratiometrically assembled with maltose binding protein (MBP) prebound to an analog of maltose covalently linked to a dyeacceptor/quencher where addition of maltose specifically displaced this analog and altered FRET in a quantitative manner.65 Moreover, the same configuration was utilized to prototype a two-step QD → dye → FRET sensor. This report was followed by an in-depth examination of the FRET processes between differentially emissive QD donors and acceptor-labeled MBP in the context of Förster theory while accounting for the presence of multiple acceptors.66 Critically, the previous two studies utilized both steady-state and timeresolved lifetime spectroscopy as part of their characterization of the underlying ET and confirm that the results did indeed match the expectations of Förster theory. Lastly, a QD donorMBP acceptor system was also reported where the acceptor was

a photochromic (PC) dye that could reversibly modulate QD PL by changing the acceptor overlap and, in turn, FRET efficiency.67 These reports would soon be joined by other groundbreaking papers on single-molecule QD FRET assays68 and in vivo FRET imaging with self-illuminating QD conjugates. 69 Interestingly, the same QD-MBP system exploited in the reports mentioned above proved to be such a robust and defined structural platform for prototyping different ET configurations that it was continuously reutilized in subsequent studies that focused on understanding QD FRET-based multiplexing,70 the utility of QDs as FRET acceptors,71 QD-protein bioconjugate structure,72 and the use of QDs as multiphoton-based FRET donors.24,73 The understanding accumulated from these initial studies provided the basis for later applications of QDs within many different FRETbased biosensors and other related configurations. Over the past decade, and beyond those researchers already mentioned, a myriad of other research groups that is far too long to list have made important contributions to understanding how QDs engage in FRET and related ET processes, and especially how QD-ET can be utilized in a biological context.28−30,74−79 Interestingly, with hindsight (and the Internet) it is also now possible to find other reports that were already foreseeing the potential for QD ET in a biological configuration. For example, despite the somewhat poor synthetic quality of CdS materials in 1992, Shumilin and coworkers were already trying to stimulate the activity of an enzyme attached to these nanocrystals by photoexciting it to putatively transfer electrons to the protein.80 Around the turn of the century, Willner started utilizing CdS QDs within biological assemblies such as DNA arrays and functionalized hydrogels.81−85 The QDs were typically illuminated in these configurations and a photocurrent was then monitored with the presence of CdS QDs typically enhancing the level of current generation to indicate the presence or absence of target. Although not exactly FRET with QDs, which would come shortly thereafter from the Willner group,86 this research still represented an important initial application of QDs and ET in a biological context. Almost all the above examples are discussed in more detail in the appropriate sections below. Lastly, as academic search engines become more powerful, we can expect that other early ventures into utilizing QDs within some ET context will also come to light and be reappreciated within a more mature field.

3. QUANTUM DOTS, SURFACE FUNCTIONALIZATION, BIOCONJUGATION, AND RELEVANT CONSIDERATIONS As mentioned, the ability of QDs to engage in FRET, FRETbased biosensing, and other forms of ET will directly depend on several inter-related materials properties. These include the type of QD material used; QD photophysical properties; QD quality; how the QD was colloidally stabilized in aqueous media and made biocompatible, which, in turn, reflects the choice of surface ligand type utilized and how the QD was modified with it; and how the bioconjugate structure was formed along with its intrinsic physicochemical properties. A review of QD ET utility with bioconjugates and within bioapplications cannot be fully appreciated without a basic understanding of these processes and their subsequent influence on the final material. Here we provide an overview built around the relevant concepts. As also pointed out within each subsection, there are many more focused and in-depth reviews concerning each 540


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of these areas that are available to the interested reader, and some of these reviews are cited for initial reference. 3.1. Quantum Dot Materials

The origins and initial driving interest in colloidal semiconductor QDs are very succinctly described by Alivisatos in a 2008 Nano Focus article recognizing the contributions of Louis Brus to this endeavor, following his award of one of the first Kavli Prizes in Nanoscience.57 Since then, many contributions have dramatically improved the synthesis, available spectral properties, overall quality, and especially the understanding of these materials. The focus in this section is not to review this area but rather to provide the reader with sufficient knowledge about certain key concepts so that they can appreciate the importance of the materials origins and their properties in the context of QDs and ET. Information on QD synthesis, function, properties, and applications can be found in some of the many informative review articles available on this subject.87−102 One of the key QD properties in the current context is that of exciton formation. In this vein, the interested reader is also referred to more detailed accounts of QD exciton structure and dynamics,103 exciton electronic coupling,104 and effects of QD surface chemistry on the subsequent optical processes.105 Since the description of what can be considered the first relatively facile synthesis of colloidal cadmium chalcogenide QDs was reported,106 these materials have attracted significant attention primarily due to their unique photophysical properties which, as briefly described above in Section 1, endow them with qualities that are far superior to conventional organic fluorophores for many applications. Foremost, as the particle’s physical size is reduced below or close to the bulk exciton Bohr diameter of the given semiconductor material(s), the band gap increases with decreasing size and becomes discrete. Due to this quantum size effect, QD PL can be continuously tuned by the size of core material. To date, a variety of semiconducting materials have been studied to synthesize QDs and their utility has been explored across many biological applications. Some of the more common representative semiconducting materials utilized in biological applications of QDs along with their PL tuning ranges are shown in Figure 3. Among these materials, the series of cadmium chalcogenide QDs (CdS, CdSe, CdTe) have undergone the most concerted development over the past decades due to the cumulative set of properties that they provide: (i) wide spectral coverage from the visible to near-infrared (NIR) region with size-dependent PL; (ii) a narrow and symmetric PL band; (iii) high fluorescence QYs; and (iv) reasonable air stability of the core-only QDs for ease of synthesis and handling. There are not many other semiconducting materials available yet with the requisite synthesis and quality that can provide all these benefits in QD form. Within biological applications, the QDs are usually used as fluorescent probes and high QY is at or near the top of the list of requirements for efficient sensing and imaging. However, core-only QDs can still be extremely sensitive to surface modifications along with ambient and especially aqueous environments. In order to improve both the fluorescence QYs and photochemical stability of QDs for practical use, the formation of core−shell structures via an overcoating of the core with a wider band gap semiconductor material has now become a well-accepted and powerful methodology.107,108 Electronically, this shell layer confines the core excitons and passivates dangling bonds. ZnS has been

Figure 3. Approximate PL emission ranges and energy levels for representative QD materials. CuInS2 and AgInS2 are noted for achieving high QYs above 50% as well.130,133 Partially adapted from ref 17. Copyright 2011 American Chemical Society.

most commonly used as the outermost shell layer due to it having the largest band gap among the commonly used QD semiconducting materials and its relatively low toxicity. Therefore, to date, CdSe/ZnS core/shell QDs remain the most extensively utilized materials within biological applications. However, overcoating of CdSe with ZnS can sometimes lead to poor overall QYs due to the large lattice mismatch (∼12%),109 which is not ideal for eliminating interfacial strain and defects. To address this issue, use of materials with smaller lattice mismatch for CdSe including ZnSe and CdS with values of ∼6.3% and ∼3.9%, respectively,110,111 and alloyed material such as CdZnS, or the combination of both strategies, have been explored and have improved both QYs and photostability.88,92,112 Within biological applications, concern about the intrinsic toxicity of the Cd component of QDs continues, and this issue is still under vigorous debate.113−115 Overcoating Cdcontaining cores with a ZnS shell appears to be a functional remedy to prevent the heavy metals from leaching out of the core and to prevent water and biologicals from contacting the core. So far, several studies have demonstrated that CdSe/ZnS QDs prepared with organic ligands that bind to the QD surface with high affinity to prevent surface desorption and subsequent QD agglomeration/precipitation in combination with a careful choice of delivery dosage (exposure time and exposure concentration) can mitigate any serious or acute toxicity concerns in cellular studies.116−123 Pilot studies with some animal models have further observed a lack of overt toxicity over long time periods following QD dosages.124−126 Nevertheless, given the contentiousness of these issues, especially in relation to long-term or persistent experimental application and later potential medical use, alternative “lower toxicity” QD materials are also currently being explored. InP is one of the most promising alternative materials with minimal associated intrinsic toxicity, and it has been demonstrated that the PL of InP/ZnS QDs can cover a spectral range from the visible to NIR region similar to that of CdSe and CdTe QDs.127−130 However, InP core QDs are extremely sensitive to oxidative degradation, and therefore significant extra care and material handling is required before the shell overcoating. 541


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Figure 4. Schematic highlighting how QDs are made colloidally stable in aqueous by either (i) ligand exchange, (ii) encapsulation, or (iii) silica coating chemistries. The center represents an as-synthesized QD in organic solvent with its hydrophobic surface of organic ligands.

progressively improved over several years; however, PL color tuning is still limited and can be sensitive to the choice of surface coating materials. Another important point to appreciate about why Cd-based QD materials are comparatively so popular is that they are still virtually unmatched by any other type of QDs when considering their narrow emission line width ( HepG2 > A549. DAPI costaining of nuclei is shown in blue. Reproduced from ref 862. Copyright 2013 American Chemical Society.

(30)

where Eo is the potential at pH 0 and h/n is the ratio of protons and electrons, respectively. The pH dependence is highlighted in Figure 113B for both dopamine and a peptide covalently coupled to dopamine. The formal potential decreases as the pH increases, which means the catechol is more easily oxidized to the quinone form, consistent with the observed increased rate of auto-oxidation of dopamine in solution at higher pH.875 Therefore, in the presence of oxygen, the catechol form will be the dominant species under acidic conditions while the quinone form is favored under basic conditions. Benzenediols and quinones are ubiquitous in biochemistry with common examples including the aforementioned neurotransmitter dopamine along with vitamin K, coenzyme Q10, and the plant derived antioxidant catechin.876 The prevalence of these and related analogous compounds with biological relevance also make them important targets for biosensor development. With this context in mind, the photophysical interactions of these compounds (i.e. dopamine) with QDs have been investigated by many groups due to their potential biological value.297,877−879 However, it is important to note that the exact nature of how these compounds interact with QDs as either energy or CT donors or acceptors, and how this interaction is manifested in the resulting QD photophysics, has been debated in the literature. Table 3 highlights this debate by presenting a partial listing of QD sensors and related constructs

similar signal transduction principles, where the MBP was labeled with a ferrocene derivative and developed a much wider dynamic sensing range than that achieved in the ensemble.872 Thrombin detection was also achieved with an aptamer-capped PbS QD, where thrombin itself was suggested to quench the QD through CT.861 8.4. Special Case of Benzenediols

Benzenediols are organic molecules with two hydroxyl groups substituted on a benzene ring in ortho, meta, or para positions (isomers shown in Figure 112A). The ortho isomer is known as catechol. Benzenediols display fascinatingly complex redox chemistry in that they can undergo a reversible two-electron and two-proton oxidation to a quinone (Figure 112B).873 While this transformation appears to be a simple conversion of reactant to product, the reversible process involves proton-

Figure 109. (A) Schematic of a lead binding protein labeled with the Ru complex (black) and assembled to a QD. Subsequent lead binding alters the protein conformation which changes the rate of electron transfer with the QD. (B) Representative PL data from titrating such a sensor with increasing concentrations of lead. Reproduced from ref 858. Copyright 2009 American Chemical Society. 643


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Figure 110. (A) Ru complex structure as attached to the cysteine thiol and when interacting with the fatty acid palmitate. (B) Schematic showing fatty acid binding protein as a ribbon structure attached to a QD (Cys, yellow; NH3+ terminus, blue; COO terminus, black ball; water, red; palmitate, black). The Ru complex would be attached at either of the 2 cysteines highlighted in yellow. (C) Palmitate bound version. Reproduced from ref 871. Copyright 2006 American Chemical Society.

where the QD and benzenediol/quinone have been ascribed the role of either eT donor to an acceptor or, alternatively, an acceptor for an eT donor. Regardless of the debate about exact mechanism of eT, many functional biosensing configurations have been described based on the combination of QDs and benzenediols. In 2006, Gill et al. described a CdSe/CdSe/ZnS multishell QD-peptide conjugate for monitoring tyrosinase activity and thrombin hydrolytic activity, where a quinone unit was used as an electron acceptor for quenching QD (donor) PL (Figure 114).847 The water-soluble QDs were coated with MPA and coupled to a peptide containing a tyrosine unit. In the presence of oxygen and tyrosinase, the tyrosine was transformed to L3,4-dihydroxyphenylalanine (L-DOPA), which underwent further oxidation to an o-quinone. The modified quinone residues quenched the QD PL via eT allowing the assay of tyrosinase activity with an LOD of 0.2 units. The peptides also contained an amino acid sequence between the QD and tyrosine unit that was recognized by the peptidase thrombin. The addition of thrombin cleaved the peptide linker, which disrupted the proximity-driven eT from the QD to the quinone residues, restoring the QD PL providing a turn-on sensor for thrombin activity. This enzymatic reaction and quenching mechanism was also adapted for MPA-coated CdTe QDs in an anodic electrochemiluminescence format.883 As an example of a QD in the suggested role of an electron acceptor, Banerjee et al. described a QD-based fluorescent probe for the detection of glutathione.880 As shown in Figure 115, the bare CdS:Mn/ZnS QDs have a yellow-orange emission maximum centered at ∼592 nm. Surface modification with dopamine ligands via a DTC linkage resulted in the loss of QD PL (“OFF” State), which was attributed to photoinduced eT from the oxidized dopamine to the QDs. QD PL was then restored upon addition of GSH, providing a 25-fold enhancement of PL with a linear response between 0 to 10 mM GSH.

The restored QD PL was ascribed to the detachment of the dopamine ligands from the QD surface, which were no longer able to engage in eT with the QD. Nadeau and co-workers have also described eT from dopamine to QDs as being responsible for QD PL quenching in conjugates meant to function as redox sensitive intracellular probes.877 The complex photophysics and behavior of these QD-dopamine conjugates were further explored in several additional papers by Nadeau’s Group.878,881,884 However, the exact mechanism for QD PL quenching in these conjugates was never fully clarified and was further complicated by the nature of the dopamine-QD attachment chemistry, which could also result in polydopamine chain formation through Michael addition. Due to the complex redox chemistry of dopamine and the potential for the reduced and oxidized forms to exist in solution simultaneously, Medintz et al. set out to design a well-defined and controlled system where the exact nature of the dopamineQD interaction could be functionally elucidated. In doing so, QD biosensors capable of measuring cytoplasmic pH changes in cells by exploiting the pH dependent redox properties of dopamine were also assembled.297 The biosensors consisted of CdSe/ZnS QDs self-assembled with dopamine labeled peptides via metal-affinity coordination between the QD surface and the peptide’s terminal Hisn sequence (Figure 116A). The sensing mechanism relied on an increase in the oxidation rate of dopamine from a hydroquinone to a quinone by O2 under basic conditions. Under neutral to acidic conditions, the appended dopamine exists predominantly in a hydroquinone form that is a poor electron acceptor. Thus, the photoexcited QDs relax via exciton recombination resulting in a strong QD PL signal. However, under basic conditions, the QD PL is quenched by photoexcited eT from the QD to the proximal quinone acceptors, see Figure 116A. Therefore, pH controls the degree of CT between the QDs and the peptide-appended dopamine molecules, and this dependence could be utilized, in turn, to 644


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Figure 111. (A) Schematic of the charge-transfer-based multiplexing. CdSe−ZnS core−shell QDs with different emissions are mixed yielding a composite multiplex emission spectrum. Bottom: Self-assembling QD subsets with Ru−phen complex labeled peptide selectively quench their PL emission (via charge transfer). Quenching of each QD color can be further tailored by varying the number of Ru−phen peptides per QD. (B) Structures of Ru(II) polypyridyl maleimide-labeled peptide (all amino acids abbreviated by three-letter code except the labeled cysteine). Composite spectra for different mixing configurations: none (C), 510 and 555 nm QDs (D), and all (E) conjugated to Ru-phen-peptide. Measured composite, deconvoluted individual QD spectra, and a fit of the summed components are shown. Reproduced from ref 843. Copyright 2009 American Chemical Society.

particular, it provides the opportunity to couple an eT process directly to QD PL for signal transduction. Several issues still remain before such a goal can be achieved. Relevant eT processes are notoriously hard to control, let alone understand, in biological and ion-rich environments. Witness, for example, the discrepancy in how benzenediols and other quinones interact with QDs. Additionally, many components of biological environments can contribute to quenching a QD beyond eT and this will serve to confuse any easy interpretation. Despite all this, combining eT with its inherent effects on QDs can open new avenues of monitoring biological processes that are not easily visualized. A prime example of this is monitoring electric signaling in neuronal cells which is foundational to neuroscience and yet still incredibly challenging in practice.885

monitor pH. Photophysical analysis of this sophisticated system revealed that the degree of QD PL quenching increased with increasing pH and with higher ratios of peptide per QD as expected. Using standard reference dyes with pH independent PL, the QD pH biosensors showed a dynamic in vitro sensing range from pH 6.5 to 11.5. In a proof-of-concept experiment, COS-1 cells were microinjected with QD-dopamine biosensors and an internal standard and allowed to equilibrate in media at pH 6.5 (Figure 116B). The cells were then placed in an alkaline buffer at pH 11.5 supplemented with the drug nystatin to induce intracellular alkalosis. QD PL was monitored over a 1 h time period, which revealed a steady decrease in QD emission as the cytoslic pH increased. The intracellular pH at each time point could be extrapolated from the PL ratio of QD to the internal standard as compared to an in vitro calibration curve using the same materials. QD PL quenching caused by eT from the QD to the oxidized dopamine-quinone was later confirmed by Ji et al. along with providing evidence for putative eT from the dopamine (in hydroquinone form) to the QD in both the excited and ground states.879 eT to or from a QD donor/acceptor in a biological assembly or biological context represents one of the most promising areas of future development for biosensing applications. In

9. SINGLE MOLECULE QUANTUM DOT ENERGY TRANSFER STUDIES Ensemble FRET measurements only provide an average value of the EFRET, masking any heterogeneity among different FRET pairs. While sufficient to provide steady-state parameters such as an average donor−acceptor distance in homogeneous systems, the function of many biomolecules involve conforma645


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Figure 112. (A) Isomers of benzenediols. (B) Oxidation of catechol to 1,2-benzoquinone. (C) The Laviron scheme describing possible pathways and species involved in the oxidation of a substituted (-R) catechol. Panel C adapted from ref 874. Copyright 1984 Elsevier.

Figure 113. (A) Structure of dopamine. (B) A plot of formal potential (Ef) versus pH for both dopamine and a dopamine-labeled peptide, which demonstrates the linear Nernstian response. Inset: cyclic voltammograms for a dopamine-labeled peptide in different pH buffers. Reprinted by permission from Macmillan Publishers Ltd.: Nature Materials,297 copyright 2010.

Pons et al. characterized this heterogeneity by correlating the EFRET and emission spectra of immobilized QDs assembled with quencher-labeled MBP.810 As expected, EFRET closely traced the spectral overlap for individual QD donors, see Figure 117A for a representative example of how FRET varies with spectral overlap. This variation was confirmed by spectral- and time-resolved measurements of the QD PL decay. Interestingly, such an analysis is also quite useful in determining whether QD interactions with some potential acceptor are dependent on spectral overlap and arise from FRET or occur due to some other ET mechanism.322,326,356 Another source of heterogeneity arises from the QD blinking behavior. In a fundamental study, Hua et al. characterized the time evolution of single QD donor-dye acceptor assemblies immobilized on a coverslip.886 They showed that, due to the QDs high absorption cross section at short wavelengths, the QD donors could be excited without significant direct excitation of the acceptors, a feature difficult to achieve with small Stokes-shifted organic dye pairs. Indeed, the dye signal

tional changes and/or multimolecular association/dissociation. These processes often create heterogeneous populations that cannot be resolved by ensemble measurements. Single molecule FRET studies, on the other hand, readily provide information about population heterogeneity and about the dynamics of FRET parameters from single molecules. Single molecule FRET techniques have been developed mainly using organic dye pairs, but QDs again present many of the same distinct advantages including photostability, brightness, narrow spectra, and multicomponent assembly capabilities that can be harnessed at the single particle level. Single QD FRET formats present several particularities compared to those based on organic dye-based FRET. First, even a relatively monodisperse QD population is composed of QDs of slightly different sizes and compositions with emission maxima that are therefore slightly different. The emission spectrum of a QD population is then the sum of the narrower spectra of single QDs. As a result, the spectral overlap between QD donors and dye acceptors actually varies from QD to QD. 646


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Table 3. Representative QD Interactions with Benzenediols and Quinones QD (emission max)

benzenediol/ quinone

electron donor CdSe/CdS/ ZnCdS/ZnS (600 nm) CdSe/ZnS (560 nm) CdSe/ZnS (∼615 nm) CdTe (570 nm) CdTe (537 nm)

electron acceptor o-quinone residues

CdSe/ZnS (520, 550, 580 nm) CdTe (570 nm) CdSe (∼560 nm) electron acceptor CdS:Mn/ZnS (592 nm) CdSe (560, 590 nm) CdSe/ZnS (605, 560 nm) ZnO (several sizes) CdSe/ZnS (572 nm)

configuration

ref

tyrosinase and thrombin sensor

847

o-quinone residue

alkaline phosphatase sensor

378

doxorubicin

DNA, cocaine, thrombin, telomerase sensor glucose sensor dopamine sensor

848

detect pH changes

297

probe DNA-anticancer drug interactions femtosecond spectroscopy

851

dopamine

detection of glutathione

880

dopamine

redox sensitive cell labels (QD size variation in eT properties) cellular probes and photophysics photophysical analysis

877

pH sensitive probes

879

benzoquinone o-quinone (dopamine) o-quinone (dopamine) mitoxantrone 1,4-benzoquinone, 1,2naphthoquinone electron donor

dopamine catechol and naphthyl catechol dopamine

849 850

867

Figure 114. (A) Scheme showing probe for tyrosinase activity in which QD PL is quenched by charge transfer to the quinone unit. Thrombin activity is probed by measuring the increased QD PL upon scission of the peptide bond. (Bottom) (B) PL spectra of 1functionalized QDs (a) before treatment with tyrosinase, (b) after treatment with tyrosinase, and (c) after treatment with thrombin to restore QD PL. (C) Time dependent QD PL intensities that trace quenching by (a) formation of quinone with tyrosinase and (b) PL restoration after treatment with thrombin. Reproduced from ref 847. Copyright 2006 American Chemical Society.

881 882

closely followed the QD blinking behavior, with no dye signal during the QD “off” periods. In addition, the photobleaching of the dye acceptors was easily recognizable by a drop in dye intensity correlated with a recovery of the QD signal (see Figure 117B). Assuming a QD QY of unity during the QD “on” periods, this enabled a direct, absolute measure of the acceptor QYs at the single molecule level. Finally, a last source of heterogeneity comes from the large surface of QDs, to which several acceptors may be conjugated. While the average QD:dye ratio may be easily controlled during the assembly process, the exact number of dye per QD usually follows a Poisson distribution. In 2006, Pons et al. measured the acceptor/donor emission ratio of QD bioconjugates freely diffusing in solution using a confocal microscope, for different QD:dye ratios.294 The emission ratio distributions closely matched those expected from the Poisson distribution (eq 17, Section 5.1), demonstrating the heterogeneity inherent to a random assembly process (see Figure 117C). In two related studies using confocal measurements of single QDs diffusing in solution, QD populations were shown to be composed of “bright” QDs with a fixed brightness and “dark”, nonemitting, QDs.887,888 The fraction of bright QDs was correlated with, but not always equal to, the ensemble QD PL QY. It remains unclear how this dark population is related to blinking, and whether this population is static and composed of the same, fixed, nonemitting QDs, or if individual QDs in solution randomly go through long bright and dark periods. Keeping these properties in mind, several types of single QD FRET studies have been realized. In particular, single molecule FRET measurements give access to the dynamics of conforma-

tional changes at the single molecule level. In a proof-ofprinciple study, Hohng et al. assembled SAv QDs with biotinylated and dye-labeled DNA strands.889 These strands were selected to form a four arm Holliday junction known to fluctuate between a compact and an extended conformation, corresponding to high-FRET and low-FRET states. The bioconjugates were then immobilized on a biotinylated surface and observed by confocal microscopy. Anticorrelated changes in the QD and dye signals were interpreted as changes in donor−acceptor distances and FRET efficiencies. Dwell time histograms of both states were constructed from a collection of time traces. The extracted average dwell times were similar to those extracted from a dye−dye FRET pair, demonstrating that the QDs were not disturbing the junction dynamics. The authors, however, pointed out that FRET was analyzable only in a small fraction of the QDs for which the junction lay close enough to the QD surface. For the vast majority of the QDs, the surface chemistry coating and the intermediate SAv layer led to donor−acceptor distances too large to enable significant QD-to-one-acceptor FRET efficiencies. Single QD FRET setups would therefore benefit from smaller QD sizes and more compact surface chemistries, and indeed the latter is something that is being actively and continuously pursued.119,120,242,890 In addition, while the QD was more photostable, the observation time window was still limited by photobleaching of the acceptor. In another somewhat related study, Sugawa et al. conjugated small peptides directly to the QD surface, which enabled compact assemblies with ATPase enzymes.891 These conjugates were immobilized on a coverslip 647


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Figure 115. (A) Scheme of the QD glutathione probe. Bare QDs are functionalized with dopamine via a dithiocarbamate linkage leading to the “OFF” state. Glutathione (GSH) turns QD PL resulting in an “on” state. (B) QD PL spectra for the bare QD (Qdot) and quenching in dopamine functionalized QDs (QDL). (C) Treatment of QDL with GSH results in increased QD PL “on” state. Inset: Shows the linear restoration of QD PL at increasing GSH concentrations. Reproduced from ref 880. Copyright 2009 American Chemical Society.

Figure 116. (A) Schematic of a charge transfer-based QD biosensor for measuring intracellular pH. The constructs consist of QDs self-assembled with dopamine labeled peptides. Under acidic conditions QD PL is strong because the dopamine exists in a hydroquinone form, which is a poor electron acceptor. Under basic, aerobic conditions the dopamine is converted to a quinone form, which is a good electron acceptor and quenches QD PL. (B) Fluorescence images of COS-1 cells coinjected with 550 nm emitting QD-dopamine biosensors and 680 nm red-fluorescent Fluorophorex 20 nm nanospheres, FLX at 0, 30, and 60 min after alkalosis. Images show a gradual decrease in QD PL with an increase in time while the internal standard dye PL remained unchanged, allowing the determination of intracellular pH through a calibration curve using the ratio of QD:FLX PL. Reprinted by permission from Macmillan Publishers Ltd.: Nature Materials,297 copyright 2010.

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(Figure 118A). The QD and dye signals were detected by TIRF microscopy to eliminate background signals from the bulk solution. Observation of FRET-induced anticorrelated changes in QD and dye intensities enabled identification of binding and unbinding events of dye-ATP molecules to the QD-enzyme conjugate (Figure 118B). The authors were then able to construct histograms of the “FRET-on” dwell times, corresponding to the time spent by the ATP molecule on the enzyme (Figure 118C). In this work, the QD photostability provided a clear advantage as it was continuously illuminated, whereas each acceptor dye only engaged in FRET for short periods of time and their lower photostability was not a limitation. The combination of FRET and TIRF microscopy enabled working at solution concentrations of acceptor-ATP molecules up to 10 μM, which is actually a much higher concentration than used in most single molecule studies. Most of the single QD FRET nanosensors have been developed in solution, where fluorescence bursts are detected when a QD bioconjugate diffuses through a confocal detection volume. Each single conjugate is characterized by the corresponding integrated donor and acceptor intensities. As mentioned, this is commonly referred to as spFRET and histograms of the acceptor/donor emission ratios, or equivalently FRET efficiencies, are then constructed from a few hundreds or thousands of bursts (see Figure 37). Pons et al. applied this technique to a QD-based maltose sensor previously developed (see Section 5.2.1).301,887 In this assay, QDs were assembled with MBP labeled with a dye acceptor at position close to the maltose binding site. The MBP conformational change upon maltose binding does not modify E FRET significantly but decreases drastically the dye fluorescence QY. Here, the QD serves both as a FRET donor and a reference signal. Ensemble studies had shown that the addition of maltose led to a continuous decrease of the acceptor signal.301,567 Single QD studies confirmed this trend at much lower QD concentrations, even though here the sensitivity was limited by the MBP-maltose binding constant. It also brought further understanding of the sensing mechanism. Instead of a progressive, homogeneous decrease of the QY of all acceptors, the data showed two QD-MBP populations: one with high acceptor/donor emission ratios corresponding to maltose-free conjugates, and one with low emission ratios corresponding to maltose-bound conjugates. Increasing the maltose concentration in solution progressively switches QD bioconjugates from the high emission ratio to the low emission ratio populations. Compared to ensemble measurements, single molecule measurements may also increase the sensitivity of FRETbased sensors: in principle, the separate acquisition of the fluorescence signals of individual FRET pairs could enable detection of a very small number of events, which would be masked by noise in average measurements. In addition, single molecule measurements typically only require small sample volumes, pushing down the limit in terms of the minimum number of detectable molecules. QD donors possess distinct advantages for these single molecule FRET assays. They act as an antenna for their FRET partner thanks to their high absorption cross section, and their brightness enables easy single QD detection. Their narrow emission spectra enable low bleed-through into the acceptor detection channel, a feature particularly important in single molecule FRET detection where photon counts are low. They can also act as nanoconcentrators thanks to the possibility of conjugating

Figure 117. (A) Normalized emission spectra for 540 nm-QDs conjugated to 20 unlabeled MBP (plain) and 20 MBP-QXL-570 (dashed, gray); a slight blueshift in the ensemble photoemission is observed. Plot of the overlap J(λ) for MBP-QXL-570 (dashed, black), together with the FRET rate spectrum for this pair (green). Reproduced from ref.810 with permission. (B) 2D plot for the correlated PL intensities of an ET particle. The bright spot around the origin corresponds to ET of the NC (QD) blinking “off” period, while clusters A, B, and C correspond to ET of the NC blinking “on” periods at stages I, II, and III, respectively. From the slopes of lines connecting the origin and points A, B, and C, as well as the line slope of CB, the ET efficiencies of stages I, II, and III can be intuitively obtained. Reproduced from ref 886. Copyright 2014 American Chemical Society. (C) Experimental emission ratio distributions compared with fits from the Poisson distribution for 540 nm QDs conjugated with N = 0.5 MBP95C−RR per QD. The contributions from the different subpopulations (n = 0, 1, 2, ...) are plotted for each value N, along with their sum, P(η), and compared with the experimental curve. Reproduced from ref 294. Copyright 2006 American Chemical Society.

and incubated with a solution of dye-labeled ATP. When these labeled ATP molecules were processed by the enzymes, the dyes came close enough to the QD surface to induce FRET 649


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Figure 118. (A) Immobilized ATPase-conjugated QDs incubated with acceptor-labeled ATP. (B) QD (green) and acceptor (magenta) fluorescence intensity time traces showing FRET-induced anticorrelated changes. (C) Histogram of “FRET-on” dwell times reflecting the kinetics of enzyme processing. Reproduced with permission from ref 891. Copyright 2010 John Wiley and Sons.

several acceptors around a single QD donor to enhance EFRET. This effect can be especially useful to counterbalance low QDto-one-acceptor EFRET due to large separation distances. In an important example mentioned previously, Zhang et al. took benefit of these features to design a highly sensitive singleQD-based DNA sensor.68 They first mixed a biotinylated capture DNA strand, a dye-labeled reporter strand and the sample to analyze. When the target DNA strand was present, it formed a sandwiched hybrid with the capture and reporter probe. The solution was then incubated with SAv QDs, enabling the assembly of the hybridized strands on the QD surface. Fluorescence signals from single freely diffusing QD conjugates were then detected using a confocal setup and used to build an EFRET histogram. When the target was absent, this histogram was characterized by low-FRET QDs only, and virtually no high-FRET QDs. Increasing the concentration of the target DNA led to the detection of higher number of highFRET fluorescence bursts (see Figure 37). Since the background signal is very low, this assay can reach a very high sensitivity, down to 4.8 × 10−14 M of target DNA. The authors then demonstrated the application of this single QD FRET nanosensor in combination with the oligonucleotide ligation assay to efficiently detect single base mutations. Since then, this single QD-DNA-FRET platform has been applied in various assays. In particular, Zhang et al. further developed this methodology in an elegant series of reports. They demonstrated that flowing the QD-DNA solutions in a capillary caused a deformation of DNA that led to higher FRET efficiencies and sensitivities comparable to bulk measurements.892 They also used QD-dye labeled-aptamer constructs to detect cocaine, using the aptamer conformation changes when binding cocaine as a way to modulate the QD-dye FRET interactions.516 Several other reports further refined the single QD-oligonucleotide FRET platform, for example, in a multiplexed format,493,893 or in combination with ligase or polymerase amplification.494,495,893 This nanosensor platform has also been extended to other substrates such as studying RNA-peptide interactions instead of DNA hybridization.894

In a different format, single QD bioconjugates were assembled then spread on a coverslip. Fluorescence signals were detected using a wide-field illumination, preferably in a TIRF mode to limit fluorescence background from the bulk solution. A CCD camera is then typically used to acquire fluorescence signals from several tens of single QD bioconjugates at a time. Donor and acceptor emission can be separated using an image splitter and acquired simultaneously to limit photobleaching issues. Emission ratio histograms are then constructed over a few tens of frames to characterize the QD population. Compared to measurements in solution as described in the previous paragraphs, this method may be simpler to perform as it does not require the use of a confocal setup. Acquisition times are no longer limited by diffusion of the conjugates out of the detection volume, so that emission ratios can be measured with a better signal/noise ratio, when taking precautions to limit artifacts from acceptor photobleaching. Both techniques are somewhat similar in terms of acquisition speed. However, immobilizing QD-biomolecular conjugates on a surface may have detrimental effects such as protein denaturation due to nonspecific interactions with the substrate. Nonetheless, several reports used this immobilization strategy to design single QD FRET-based biosensors.403,446,895,896 In these studies, surface immobilization most often occurs after the targeted biomolecular interaction, to limit interference from the substrate. Similar to the assays described previously, the use of QDs enables easy single molecule detection thanks to their high brightness, and the assays are designed such that multiple acceptors can assemble around each QD to enhance FRET efficiencies. For example, Wang et al. designed a single QD FRET nanosensor to probe the activity of cyclic adenosine monophosphate- (cAMP)dependent PKA, which may serve as an extracellular tumor biomarker, see Figure 119.446 When active, this enzyme catalyzed the transfer of a biotinylated phosphate from BtATP to a dye-labeled peptidic substrate. The dye- and Btappended peptides can then assemble on a SAv QD and engage in FRET. Fluorescence images of single QDs on a coverslip 650


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Figure 119. Schematic Illustration of the protein kinase assay and detection of endogenous activity. (A) With γ-biotin-ATP as the phosphoryl donor, the PKA-catalyzed phosphorylation reaction incorporates the biotin entity into Cy5-labeled peptide substrate, which can assemble onto the surface of QDs through specific biotin−streptavidin interaction to form the sandwiched QD−peptide−Cy5 nanostructure. The subsequent FRET between the QD and Cy5 enables the detection of Cy5 signal. The protein kinase activity can be evaluated by the measurement of Cy5 signal. Inset shows the structure of γ-biotin-ATP. Detection of endogenous PKA activity. (B) Emission spectra of 605QD and Cy5 in response to the control group without cell lysate (olive color), the unstimulated cell lysate (green color), 10 μM forskolin (Fsk)/20 μM 3-isobutyl-1-methylxanthine (IBMX)-stimulated cell lysate (blue color), 50 μM Fsk/100 μM IBMX-stimulated cell lysate (red color), 50 μM Fsk/100 μM IBMX-stimulated cell lysate with the addition of 10 μM H-89 (black color). (C) The Cy5 counts obtained via the TIRF-based counting measurement as shown in panel A. The total protein concentration of HeLa cell lysates used in each experiment is 10 μg/mL. Error bars show the standard deviation of three experiments. Reproduced from ref 446. Copyright 2015 American Chemical Society.

induced cleaving of a dual dye- and Bt-labeled DNA strand, which led to reduced QD FRET efficiencies.895 Overall, single QD FRET formats can be both useful and informative; however, they still appear to be quite underused in actual practice. This may be due in part to both the complexity of the instrumentation required along with the requisite skill levels needed to put it into practice. It is probable that use of single QD FRET formats will only expand as these materials become more widespread and accepted in the fluorescence community, the methods for achieving discretely controlled QD-based donor−acceptor bioconjugate structures improve, and some of the necessary instrumentation is simplified and made less costly. Lastly, the primary driving factor to increase this application will be the need to implement it. Highthroughput pharmaceutical screening of large drug libraries may represent one such potential area as they inherently require minimal use of reagents.

were acquired under QD donor excitation and the number of spots in the acceptor channel was measured. Thanks to the limited QD bleed-through in the acceptor channel and TIRF acquisition, no acceptor fluorescence was detected in the absence of PKA. This very low background then enabled very sensitive detection of small PKA quantities. This sensor was then successfully demonstrated on cell lysates to probe intracellular PKA activity. A similar assay was developed to probe telomerase activity, where telomerase induced the addition of a dye-labeled nucleotide on a reporter probe, which was hybridized with a biotinylated capture probe and assembled on SAv QDs.896 In contrast to these FRET-on assays, other assays have been developed in a FRET-off format. For example, Long et al. probed the activity of rennin using a dual dye- and Bt-peptide substrate, which assembled on QDs and induced FRET in the absence of renin. This peptide was cleaved in the presence of renin, which reduced the number and intensity of FRET-active single QDs.403 Similarly, Zeng et al. probed point mutations in microRNA using rolling circle amplification. The presence of the amplified RNA target 651


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Figure 120. Ratiometric FRET probe for Zn2+ detection. Dual-emission silica-coated QD mixture (512- and 572 nm emitting) employing a porphine-based chelator (TSPP) as a receptor for cytosolic Zn2+ ions. In the absence of Zn2+, FRET from the 512 nm QDs to the TSPP dominates (FRET-1) and emission in the 572 nm window is observed. In the presence of Zn2+, FRET between the 572 nm QDs and Zn2+-loaded TSPP dominates (FRET-2) and emission is observed in the 512 nm window. Reproduced from ref 899. Copyright 2015 American Chemical Society.

Figure 121. (A) Schematic highlighting the assembly of the FRET-based Ca2+ biosensor. (B) Local Ca2+ detection. Traces show simultaneously acquired transients in distinct spots from the QD FRET data. (C) Pseudocolor-overlay of spot positions on the cell outline. (D) Repetitive simulation evokes reversible Ca2+ transients with superimposed traces, recorded at a same point. Reproduced from ref 901. Copyright 2014 American Chemical Society.

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of influxing Ca2+. To address these limitations, Zamaleeva et al. devised a FRET-based construct comprised of a commercial 565 nm-emitting QD as a donor and CaRuby, a red-emitting Ca2+-indicator, as an acceptor, see Figure 121.901 The CaRuby dye had already undergone preliminary FRET testing with QDs previously.290 Cytosolic delivery of the sensor to baby hamster kidney cells expressing a N-methyl-D-aspartate receptor construct was achieved by appending to the QD surface 5− 10 copies of a cell-penetrating peptide (H11) derived from the scorpion toxin hadrucalcin, which targets the intracellular ryanodine receptor. Appending of a PEG modified-CaRuby to the QD did not abrogate the sensitivity of the Ca2+-indicator and detection of Ca2+ transients spanning the range of 3−20 μM was demonstrated. Importantly, whereas most previously reported ionic nanobiosensors required direct microinjection into the cytosol, the H11 peptide on the QD-CaRuby sensor facilitated efficient endoosmolytic delivery. Here, the sensors adopted a pointillistic distribution which allowed for the localized readout of Ca2+ transient concentration, particularly in the proximity of the plasma membrane. The detection and quantification of mercury, a highly toxic heavy metal that is prone to accumulation in biological tissues, is typically performed using bulky, expensive analytical instrumentation in dedicated facilities, and reports of sensitive methods for in situ mercury detection are still quite limited. Further, fluorescence-based methods for real-time, intracellular Hg2+ detection are typically single, “signal-on” fluorophore systems in which the sensitivity can be influenced by the concentration of the probe molecule. Multiple QD-dye FRET systems have been described to try to address these shortcomings.584,586,864,902 The Wang group reported on a system based on an N-acetyl-L-cysteine-functionalized QD donor (508 nm emission) engaged with a metal-responsive Rhodamine 6G (Rh6G) acceptor (554 nm emission).903 In this system, the nonfluorescent Rh6G derivative was first mixed with the Hg2+-containing sample to generate a fluorescenceemitting Rh6G-Hg2+ complex via desulfurization and formation of the fluorescent amide-containing form of the dye. Subsequent addition of the QD donor to the system resulted in solution-based FRET between the QD and metal-bound Rh6G acceptor. Using this system, Hg2+ was detected down to ∼100 μg/L (∼500 μM) in live HeLa cells. QD-based FRET methods for the detection of other ion species have also been reported. The Wong group, for example, have developed a crown ether-based sensor for sodium ions,593 and monothiolated anion receptor-based sensors for chloride ions.904 These ion species are involved in maintaining the cell’s resting membrane potential (and restoring it after depolarization events) as well as regular homeostasis and subcellular organelle functions. In both of these systems, the targeted analyte was detected at relevant physiological concentrations with minimal interference from competing ion species. In contrast to ions, sensing dissolved gases intracellularly and in tissues can be extremely challenging experimentally. Fluorescence-based imaging of the dynamics of oxygen consumption in real-time has been critically hampered due to the reliance on organic dyes. Ingram et al. attempted to overcome this hurdle by using a 590 nm-emitting QD donor coupled with an oxygen-sensitive platinum(II)-octaethylporphine ketone acceptor engaged in FRET.905 Entrapment of the FRET complex within a polymer matrix (polyvinyl chloride) and subsequent deposition of the sensor-loaded polymer onto glass substrates or optodes enabled the visualization of [O2]

10. CELLULAR-BASED SENSING The assessment of cellular homeostasis or pathogenesis, particularly in real-time, is critical for ascertaining cell health. Of the various NP formulations currently under development, QDs have emerged as one of the more popular sensing platforms for the in situ sensing/interrogation of cellular processes and molecular indicators of cell health.18,22,369,372 In this Section, we review some of the more recently demonstrated applications of QDs engaged in either FRET or CT for the real-time monitoring of targeted processes at various cellular locations. For clarity, we divide our discussion of this topic between the sensing of targets and processes that occur primarily within the confines of the cytosol (intracellular), and those that occur (or at least are initiated) at the cell’s plasma membrane/extracellular space. 10.1. Cytosolic Targets and Intracellular Molecular Assembly

10.1.1. Ions/Cofactors. The intracellular concentration of various ions is a critical modulator of cellular homeostasis and numerous reports have documented QD-based FRET sensing schemes for the detection/measurement of a range of ion species. After iron, zinc is the second most abundant transition metal in the body where it exists primarily in chelated/ sequestered form (e.g., localized within the synaptic vesicles of excitatory nerve terminals).897 On release, however, free Zn2+ alters the behavior of several ion channels and receptors.898 To provide insight into some of these processes, Wu et al. devised a dual QD FRET ratiometric approach based on Zn2+ ion chelation by pendent meso-tetra(4-sulfonatophenyl)porphine dihydrochloride (TSPP) moieties (Figure 120).899 The TSPP served a dual role as both a Zn2+ chelator and as a tunable acceptor/QD quencher based on its Zn2+-dependent shifts in absorption spectra. Two populations of QDs, with emissions at 512 nm (green) and 572 nm (yellow), were overcoated with a silica layer (which acted as a spacer) and the TSPP chelator was conjugated to the silica coating. In the absence of Zn2+ions, FRET occurred from the 512 QD to TSPP, which resulted in a two-channel signal (low 512 nm/high 572 nm). In the presence of Zn2+, ions bound into the TSPP binding pocket and shifted the absorbance spectra of the TSPP so it now served as an acceptor for the 572 nm QDs and shifted the FRET signal readout to high 512 emission/low 572 emission. This sensor scheme detected Zn2+ ions in a linear fashion over the range of 0.3−6.0 μM and within a pH window of 5.0−8.0. The ratiometric sensor also underwent preliminary testing in live HCT116 human colon carcinoma cells following passive uptake, where it was able to confirm that the cells had been treated with 6.0 μM ZnCl2. The authors did note, however, that given the slow chelation kinetics of the TSPP chelator, additional catalysts (imidazole, Cd2+) had to be included in the delivered sensor mix and served to effectively shorten the response time of the sensor to ∼30 min. Ruedas-Rama also targeted intracellular Zn2+ detection with a turn-on QD sensor that utilized photoinduced eT between an azacycle receptor group and the QDs.900 Calcium is an ion that plays a critical role as a ubiquitous second messenger in multiple intracellular signaling pathways. The rapid, fluorescence-based detection of local Ca2+ ion concentrations has long been a challenge because of the low signal-to-noise of the available indicating sensors and their poor sensor diffusion kinetics that results in the localization of only a few sensor molecules proximal to the near-membrane volume 653


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Figure 122. (A) Scheme of FRET transfer between QDs and the phosphorescence quenching oxygen dye PtOEPK. (B) Excitation of an optode thin film using two-photon excitation at varying QD:phosphorescent quenching dye (OSD) ratios. 860 nm excitation and a mass ratio of 1:3 of QD:PtOEPK produced a balanced emission intensity ratio at 590 and 757 nm. (C) Shown is the real-time imaging of [O2] in distinct brain regions showing changes at point x primarily localized within the CA1 prior to aminopyridine-induced seizure onset. Adapted from ref 905. Copyright 2013 Elsevier.

alkalosis. See Section 8.4 for a more thorough description of this sensor. More recently Bao’s group developed a QDmOrange fluorescent protein donor−acceptor FRET pair in which the spectral changes in the covalently attached acceptor’s absorption and emission spectra as a function of pH translated to pH-dependent changes in the EFRET of the sensing construct.282 Specifically, the molar extinction coefficient of the mOrange acceptor is maximized when the pH approaches the protein’s pKa. Compared to the most commonly used pHsensitive fluorophore (BCECF), which exhibited a ∼ 5-fold change in signal over the pH range 5−10, the QD-mOrange sensor displayed a remarkable ∼16-fold change in signal over this same range while concomitantly offering significantly enhanced photostability due to its indirect excitation by the QD; the latter benefit is something that is seen repeatedly in the literature when using QD donors. Modification of the protein acceptor to express a terminal polyarginine motif enabled the real-time measurement of the acidification of endosomal compartments over a 2 h time window in HeLa cells. See Section 5.2.7.2 for more discussion of this construct. 10.1.3. Intracellular Assembly and Disassembly. QDs have already been used in multiple reports for the real-time imaging and tracking of the assembly/disassembly of macromolecular constituents or stability studies of multicomponent molecular architectures. Many of these examples have focused on the complex interplay of therapeutic nucleic acids (pDNA or siRNA) that are delivered using the QD as both a scaffold and a sensing moiety to track delivery progress and fate over time. Still other demonstrations have examined intracellular assembly of QDs that are targeted to interact with specific intracellular proteins. While the examples discussed here by no means

gradients across distinct regions of rat hippocampal brain slices during onset of drug-induced seizure, see Figure 122. The cofactor 1,4-nicotinamide adenine dinucleotide phosphate, NAD(P)+, is another small molecule that plays a critical role in biological transformations in the cell and multiple redox enzymes utilize it as an electron transport shuttle to carry out their functions. As mentioned earlier (Section 5.2.3.4), a QDbased sensing system utilizing Nile Blue as an electron mediator for the oxidation of NAD(P)H cofactors was detailed by Freeman et al. where 635 nm-emitting CdSe/ZnS QDs were decorated with BSA. Nile Blue, noncovalently associated with the BSA, quenched the QD when in its oxidized form and upon reduction to NAD+ during transformations, resulted in QD reemission. This sensing platform was used to visualize the activity of NAD+-dependent AlcDH activity when injected into HeLa cells.166 10.1.2. pH. Given its critical role and the possibility for loss of homeostatis if not carefully controlled, intracellular pH is tightly and spatially regulated by ion transport systems and the high buffering capacity of the cytosol. Several groups have reported on QD-based methods for measuring cellular pH. In a sensing scheme based on CT, Medintz et al. designed a pHresponsive QD-dopamine peptide hybrid based on the redox properties of the appended dopamine ligand. Here, 550 nmemitting QDs decorated with peptides displaying a covalently attached dopamine were injected into the cytosol of COS-1 cells. eT from the excited state QD to the quinone form of dopamine resulted in the quenching of QD PL in a pHdependent manner.297 This construct visually reported realtime discrete changes in pH over the range ∼6.6−11.5 over a 1 h time period as the cells were subjected to drug-induced 654


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Figure 123. Monitoring plasmid DNA decondensation in nuclear subdomains. Dye-labeled biotinylated plasmid DNA complexed with streptavidinQDs served as donor−acceptor system to ascertain the preferred unpacking location of the complexes. QD re-emission (green) denoted the efficient unpacking of plasmid DNA in the euchromatin subdomains in the nucleus. Adapted from ref 906 with permission of Oxford University Press.

encompass all of the work done in this area, they are meant to give a flavor for the types of assembly and tracking applications in which QDs have played a key role. Multiple examples appear in the literature of the use of QDdye FRET for visualizing the time-resolved cellular fate of pDNA-nonviral vector (e.g., lipid or polymer) nanocomplexes. In one of the first reports, Ho et al. used a biotinylated plasmid encoding EGFP that was labeled with SAv-conjugated 605 nmemitting QDs and subsequently complexed with Cy5-labeled chitosan (as the nonviral delivery agent).505 ET between the QD-dye pair tracked the dissociation of the plasmid-chitosan nanoplexes in HEK 293 cells over a 72 h period as the plasmid escaped the endosome, translocated to the nucleus and resulted in cellular GFP expression. Shaheen et al. citing the important role of pDNA decondensation in obtaining efficient transient gene expression, devised a QD-based FRET scheme to visualize the preferential unpacking of pDNA in specific nuclear subdomains (euchromatin domains).906 Their multifunctional system was comprised of SAv-QD donors appended with dyelabeled, biotinylated pDNA. The complexes were encapsulated in multilamellar liposomes containing endosome- and nuclearspecific fusogenic lipids along with polyrotaxane, which is a custom, disulfide-containing polycationic condensing agent. QD PL increases due to loss of acceptor proximity denoted the efficient unpacking of pDNA that occurred preferentially in the euchromatin subdomains in the nucleus (Figure 123).906 Wu et al. used a FRET approach to conduct a comparative analysis of the uptake and dissociation kinetics of lipoplexed- (lipid) versus polyplexed (polymer)-oligonucleotides. In KB cells (human oral carcinoma; subline of HeLa), it was shown that while the efficiency of cellular uptake of the 605 nm QD-Cy5-labeled oligo complexes was comparable, the QD-oligo dissociation rate in the polyethylenimine (PEI) polymer was slightly faster than the lipid complexes.907 This faster dissociation rate was attributed to the electrostatic nature of the oligo-polymer association compared to the encapsulation of the QD-oligo complexes in the lipoplexes. The net result was more efficient delivery of the QD-oligo complexes to the cytosol with concomitant rapid dissociation of the QD-oligo complexes.

The intracellular fate and delivery kinetics of therapeutic siRNA has also become of wide interest for its potential in modulating the expression/activity of targeted genes. Lee et al. focused on the quantitative delivery and unpacking of siRNAs targeting the knockout of the VEGF gene as involved in angiogenesis.908 The latter is the process of new blood vessel formation that occurs normally under physiological conditions during tissue and organ development, but can also occur when aberrant vessel growth is stimulated and recruited for tumor progression and extravasation. In their work, 625 nm-emitting QDs were conjugated to PEI via EDC chemistry and electrostatically assembled with Cy5-labeled VEGF siRNA and Cy5-labeled siRNA with PEI. Cellular uptake of the ensemble was driven by the addition of a protein transduction domain peptide from the human Hph-1 transcriptional factor. In PC-3 cells (prostate-derived adenocarcinoma), the timeresolved dissociation of the dye-labeled siRNA was tracked over a 6 h period. Confocal analysis showed clear FRET signal from the QD donor to the dye acceptor as the complexes were bound to the plasma membrane at 30 min and this signal diminished in a time-dependent fashion, resulting in QD donor PL increases as the complexes escaped the endosome to the cytosol. These observations were confirmed by fluorescenceassisted cell sorting (FACS) analysis. Introduction of these complexes to cells resulted in ∼6-fold knockdown of VEGF protein production relative to control.908 In work that focused on monitoring of assembly processes in real-time (as opposed to disassembly), Medintz et al. demonstrated the ability to generate QD-fluorescent protein assemblies in situ within the cytosol of living cells. This work built upon previous work in which metal-affinity interactions were used to assemble, and subsequently deliver via endocytosis, QD-YFP conjugates wherein the YFP was selfassembled to the QD surface through a terminal His6-tract.909 Later this assembly scheme was extended to realize the assembly of a His6-bearing mCherry acceptor protein as it conjugated noncovalently to the surface of a Ni2+-loaded polymer-coated commercial QD preparation.216 COS-1 monkey kidney cells expressing His6-mCherry in the cytosol were microinjected with the donor QDs and the FRET was 655


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monitored in real-time over a 6 h experimental window. Notably, minimal reduction in EFRET was observed over this time period demonstrating the intracellular cytosolic stability of the formed complex. Further, the QD-sensitized excitation of the mCherry acceptor in a FRET configuration resulted in an enhanced photostability of the mCherry acceptor. This work was recently continued with an examination of how QD surface ligands affect intracellular assembly along with how the assembly process can be localized to a particular subcellular site such as the inner leaflet of the membrane.910 A similar FRET-mediated photostability enhancement of a QD-sensitized membrane labeling acceptor dye (in this case an organic dye) was noted in studies of targeted QD delivery to the plasma membrane (see Section 10.2).118 10.2. Plasma Membrane and Related Extracellular Processes

10.2.1. Membrane Dynamics. The plasma membrane stands as the cell’s first line of defense against the extracellular environment and the regular turnover of membrane components within it (e.g., phospholipids, proteins, sugars) is critical not only to maintaining cell health, but can function as a sentinel marker for the onset of disease.911 For example, the socalled “phospholipid effect”, initially characterized by changes in the turnover of membrane phospholipids on stimulation of exocrine tissues, has subsequently been observed in a variety of tissues and has been shown to be the result of changes in membrane composition of phosphatidylinositol.912 Multiple examples of QD-based FRET sensing schemes geared to detecting the behavior of key membrane components have been described to date and we review some representative examples that highlight what these sensors can provide in this configuration. The Mulder group fabricated lipoprotein-based NPs comprised of a 620 nm QD core and a Cy5.5-conjugated lipidic coating as a platform for visualizing the transition of labeled-lipids to/from the plasma membrane as evidenced by the resulting changes in EFRET.913 The EFRET of the system could be tightly controlled by modulating the ratio of dyelabeled lipid in the NP assembly. Initial testing outside of cells demonstrated the quantitative exchange of lipids between highdensity lipoprotein containing QD micelles (HDL-QDs) and Cy5.5-lipid labeled micelles. The inherent role of HDL is to bind and remove excess cholesterol/lipids from the membranes of peripheral cells, particularly from lipid-rich macrophages. When HDL-QDs doped with Cy5.5 lipids were applied to THP-1 monocytic cells, they observed the real-time transitioning of the Cy5.5-lipids back and forth between the NPs and the plasma membrane, see Figure 124. QD FRET can also be used to probe dye-labels already present across the cellular membrane. As part of an extended structure−function-activity study of a peptide sequence (peptide JB577) that facilitated the endosomal uptake and escape of a range of NMs including QDs, Boeneman et al. identified a variant motif (peptide JB858) that appended QDs onto the outer leaflet of the plasma membrane with what appeared to be long-term residence (>48 h) with minimally observed cytotoxicity.118 The membraneappended QD was shown to be an efficient FRET sensitizer to two different intramembrane-labeling dyes. In one of the demonstrated examples, the plasma membrane in PC12-Adh rat pheochromocytoma cells was colabeled with 550 nm QDJB858 conjugates and a rhodamine-labeled phosphoethanolamine (Rhod-PE) lipid and subsequent direct excitation or

Figure 124. QD-lipoprotein micelles for monitoring lipid transition into cell plasma membranes. (Top) 620 nm-emitting QDs assembled into micelles doped with Cy5.5-labeled phospholipids and apolipoprotein (ApoA-1) are used to monitor the transfer of phospholipids from the QD-micelle NP into the plasma membrane of THP-1 monocytes. The real-time decrease in FRET efficiency as Cy5.5-lipid desorbs from the NP reports the transition. (Bottom) Fluorescence image of THP-1 cells showing the unquenched emission from Cy5.5 lipids that have transitioned into the plasma membrane. Scale bar, 20 μm. Adapted from ref 913. Copyright 2010 American Chemical Society.

FRET excitation imaging of the Rhod-PE showed an enhanced photostability of the sensitized Rhod-PE emission with no leakage of the donor signal into the acceptor channel (Figure 125). Just as important as understanding the dynamics of phospholipid behavior in the plasma membrane, it is equally important to probe and ascertain the function of integral and peripheral membrane proteins. In an example that epitomizes this specific focus, Kang et al. devised a FRET-based system to understand the geographical colocalization of two membrane proteins involved in several cancers.914 In their system, the proteins nucleolin and integrin αvβ3 were each appended with a donor and acceptor. Nucleolin was labeled with a Cy3-labeled aptamer specific for that protein and the integrin was labeled with a 520 nm QD donor by conjugation to an RGD peptide, which subsequently drove the labeling of the integrins with the QDs. Single cell imaging was performed in HeLa cells and a direct comparison was made between the quantification of the degree of colocalization of the two proteins as determined by either direct imaging of the dye and QD PL and overlay or by EFRET measurements. Across the same linescan in the same cell, EFRET-based imaging clearly showed a higher degree of protein colocalization than when the channels were collected separately by direct excitation of each fluorophore and overlaid. In an elegant example of trying to extract colocalization information, the Meissner group designed a cantilever-based system to monitor the formation of focal adhesions/contacts in real-time by probing the surface of HeLa cells with a QD-appended AFM tip.915 QDs, coated with fibronectin were covalently attached to the AFM tip and the probe tip was brought into proximity of 656


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Figure 125. (A) Plasma membrane of PC12-Adh cells was labeled sequentially with 125 nM 550 nm QD-JB858 conjugates (1 h incubation) followed by 20 min incubation with 10 μM Rhod-PE. The dye-FRET panel shows sensitization of the dye by the QD donor (R0 ≈ 5.2 nm). (B) Comparison of the photostability of membrane resident QDs, Lissamine Rhodamine B phosphoethanolamine, and the QD-Rhodamine bilabeled cells. Sensitization circumvents direct dye photobleaching. Size bar = 50 μm. Adapted from ref 118. Copyright 2013 American Chemical Society.

the surface of cells expressing αv integrins with monomeric red fluorescent proteins (RFP) fused to their carboxyl termini. Thus, the RFP was located on the inner leaflet of the cell’s plasma membrane while the FN-coated QD probed the integrin binding site. Photobleaching of the RFP acceptor was used to demonstrate successful FRET by capturing subsequent increases in the QD donor PL. Focal adhesions, which are clusters of integrin proteins collected into tight groups on the plasma membrane, were formed within 20 min of bringing the QD-decorated probe tip into contact with the cell surface. These results suggest that this technique is an important and viable approach with which to interrogate the function of integrins and membrane proteins within the lipid bilayer and their interaction with the extracellular environment. 10.2.2. Receptor−Ligand Interaction and Endocytosis. Just as there is a critical need for probes to study membrane dynamics and protein function within the plasma membrane bilayer, it is also necessary to understand in fine detail the balance between receptor−ligand interactions and endocytosis. From the earliest seminal studies showing the ability to label and track receptors with QDs without abrogating their native function (see ref.916 and references therein), there has been a rich and steady growth in the implementation of QDs for FRET-based imaging of receptor−ligand interactions and endocytosis. Early pioneering work in this area was performed by the Barossa Group studying the organization, distribution and endocytosis activity of the transferrin/transferrin receptor/ ligand system.917 Their approach consisted of conjugating the transferrin ligand to either QDs or the cognate AlexaFluor dye acceptor. Upon incubation of the labeled transferrin proteins

with cells, QD-dye FRET was used to track the ligand uptake efficiency into endosomal compartments and to track the transport process along the endolysomal system. These enabling studies also demonstrated the inherent benefit of using QDs as FRET donors in such systems, which can reduce the burden of spectral deconvolution and data processing that is required when using organic dye pair FRET systems.918 The same group extended their system to perform comparative analysis of the endocytic pathway in normal versus cancer cells.919,920 Analogous studies have also shown the benefits of QD-dye FRET pair systems in studying the endocytic pathway using two-photon fluorescence microscopy.24 Still other FRET-based approaches have used QDs to monitor the important process of receptor dimerization during cell activation. Kawashima et al., for example, used single molecule fluorescence imaging and FRET observations of cellsignaling propagation by EGFRs coupled to QDs.447 The authors set out to better understand the lateral propagation of EGFR activation through EGF ligand binding in the extracellular domain which results in EGFR dimer formation, which is, in turn, integral to subsequent regulation of cell signaling, growth, and proliferation. Critically, when this activity is left unchecked by a cell, it can promote the uncontrolled proliferation of different cancers. Activation of EGFRs in live human A431 ovarian epidermoid carcinoma cells was carried out by binding QDs to recombinantly expressed EGF. Activation and colocalization of EGFR and EGF was confirmed using FRET with a Cy5-labeled anti-EGFR monoclonal antibody (Ab11), see Figure 126. The FRET generated from the association of QD-EGF with Cy5-Ab11 through EGFR was 657


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Figure 126. (A) Florescence image of living A431 cells activated with 2 nM QD-EGF. (B) Fluorescence and (C) FRET images of an A431 cell labeled with Cy5-Ab11 and activated with 0.5 nM QD-EGF. Histograms of fluorescence intensity from A431 cells activated with (D) 2 nM and (E) 0.5 nM QD-EGF. (F) Histogram of fluorescence intensity from QD-EGF single molecules without cells. The solid curves in (D) and (E) are a guide for the eyes. The statistical distributions of heterodimers, homodimers and oligomers of EGFR were estimated from Gaussian fits. Reproduced with permission from ref 447. Copyright 2010 John Wiley and Sons.

further probed using time-resolved fluorescent spectroscopy. A change in the QDs PL lifetime from ∼7 ns to ∼3 ns was observed in the absence (unbound) and presence of Cy5-Ab11 (bound), respectively. The decrease in the QD fluorescence lifetime was accompanied by sensitized-fluorescent emission from Cy5. This study provided strong evidence that the lateral propagation of EGFR takes place through continuous exchange of monomeric EGFR units. To investigate the reversible association and dissociation of EGFR in A431 cells, FRET was also used with two complexes, a QD-labeled EGF-EGFR unit and a Cy5-labeled Ab11-EGFR unit. Microscopy revealed propagating FRET spots, suggesting either continuous exchange of the two units to form QD-EGF-EGFR/EGFRAb11-Cy5 heterodimers or possibly the lateral diffusion of the associated heterodimers. This work in living cells suggested that lateral activation of EGFR propagated through transient dimerization of a heterodimer [EGF(EGFR)2] with a predimer [(EFGR)2], thus furthering the understanding of EGFRmediated cell signaling and helping elucidate potential EGFRtargeted inhibitors. Finally, in an example that illustrates the use of the endocytic pathway while integrating multiple targeting/sensing functionalities on a single QD FRET platform, Wei et al. designed a combinatorial QD-oligonucleotide structure for the dual

membrane targeting/endocytosis coupled with subsequent cytosolic neutralization of mRNA in HeLa cells.491 The construct consisted of a central 610 nm-emitting QD core associated with a noncovalently complexed heterobifunctional oligonucleotide (Figure 127). See also Section 5.2.4.1.2 and Figure 36B for specifics on how this sensor functioned mechanistically. Assembly of the oligo to the QD was driven by the phosphorothioate domain in the oligo center, which facilitated dative bonding of the oligo to the Cd2+-containing QD core while each end of the oligo was designed to serve separate functions. At one end was a covalently attached aptamer (AS1411) directed against the cell surface protein nucleolin, which is overexpressed in certain cancer cells. The other end of the core oligo contained an antisense DNA oligo specific for survivin mRNA, a transcript like the nucleolin protein that is upregulated in cancerous HeLa cells. During delivery and uptake, the antisense oligo is blocked by hybridization to a complementary Cy5-labeled DNA strand. FRET between the QD donor and Cy5 acceptor served as a reporter for the DNA melting and exposure of the antisense oligo for target binding. The binding of the assembled complex to nucleolin via the AS1411 aptamer mediated endocytosis followed by leakage of the complexes to the cytosol where the antisurvivin antisense oligo could then target its mRNA 658


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Figure 127. Heterobivalent QD-oligo probe for dual targeting mRNA in cancer cells. (Top) A central 610 nm emitting QD core is assembled with bifunctional oligo. One end of the oligo bears an aptamer against cell surface nucleolin protein for pinocytosis and cytosolic delivery of the nanoprobe. The other end is an antisurviving mRNA antisense DNA oligo that is hybridized to a Cy5-labeled complementary oligo to monitor the hybridized state of the antisense probe. (Bottom) FRET confirms dissociation of the Cy5-labeled “blocking” oligo. The Cy5 (FRET) channel shows significantly less signal when paired against the QD channel indicating a decrease in the amount of Cy5-labeled oligo in the complex. Comparison of the direct Cy5 excitation with the QD direct emission also shows less Cy5 signal compared to QD signal. Reproduced with permission from ref 491. Copyright 2014 John Wiley and Sons.

was then tailored for NIR imaging by replacing the donor and acceptor in the ensemble with a 720 nm-emitting CdTeS QD and the NIR-emitting dye ICG-Der-02. Intratumoral injection into an MDA-MB-231 mouse tumor model showed significant MMP2 activity over a 3 h period in vivo that corresponded to tumor-secreted MMP2, see Figure 128. The Wang group developed a combinatorial probe for the MMP, MT1 (MT1MMP).921 Here, a “bent peptide” approach was used to append an MT1-MMP substrate peptide onto the surface of 525 nmemitting QDs. The bend was induced by a peptide design that placed an Arg9 (positive charge) and Glu8 (negative charge) motif at opposite ends of the peptide. A terminal Cy3 acceptor was held proximal to the QD donor via electrostatic interactions (FRET “on” in the absence of protease). In the middle of the peptide was an RGD motif for plasma membrane binding and an AHLR substrate sequence recognized by the MT1-MMP. Upon protease cleavage, both the QD signal increased (FRET “off”) and the exposed Arg9 motif facilitated cellular uptake of the now-emitting QD donor, thus giving a biphasic readout of MT1-MMP activity (QD donor re-emission coupled with cellular internalization). This represents an exciting example of the progress toward developing smart multifunctional NMs where one targeted biological event (protease activity) results in two subsequent NM or cellular events−namely, signal transduction and cellular uptake.45 Although only a limited cross-section of what has been reported,369,372 these examples still reflect the strong potential that QD-based FRET has for intracellular sensing.

complement. Initial tests showed the specificity of the complex for nucleolin-expressing cells as the QD signal could be seen preferentially bound to HeLa cells in direct comparison to HEK 293 cells (nucleolin-negative). Further characterization showed the time-dependent decrease in both QD-Cy5 FRET and colocalization as evidence of the melting and targeting of the antisense oligo to the survivin mRNA. 10.2.3. Activity of Secreted Proteases. One of the more important biological processes associated with the plasma membrane is the secretion of active molecules and, among these entities, proteases represent one of the larger classes of secreted “active” biologicals. Proteases are involved both in the regular maintenance of cell health as well as the progression of disease, including many cancers, and are the target of significant drug development efforts. In an early example of the use of QD FRET for cellular protease sensing, Shi et al. designed FRET probes for visualizing the activity of MMPs that were secreted into the culture medium of cancer cell lines.392 545 nmemitting QDs decorated with Rh-labeled RGD/substrate peptides were incubated in the media of a breast cancer cell line (HTB 126) and a control cell line (HT125); only QD donor re-emission was observed in the HTB 126 line that was expressing active MMPs.392 More recently Li et al. devised a QD-peptide construct comprising a 535 nm-emitting CdTe QD donor and an MMP2-specific peptide (GPLGVRGKGG) coupled to a rhodamine B acceptor.400 This ensemble was first functionally tested in MDA-MB-231 breast cancer cells to show the specific sensor changes induced by proteolytic cleavage and increasing donor emission in the green channel. This system 659


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important issue that is intertwined with the previous is that of getting the QD-FRET sensors to the exact intracellular site required for a particular application. In almost all cases, this requires both escaping the endolysosomal system along with accessing subcellular organelles in many cases. Methods for robustly accomplishing this in a repeatable fashion with any type of NP-bioconjugate are still lacking and keenly required. For more in-depth discussion on this important issue, the interested reader is referred to several reviews and other related papers.116,118,157,369,916,923−928 A directly related focus area to this Section that may also see concerted expansion in the near future is that of implementing QD-based single molecule FRET sensors intracellularly. Here, the delivery and targeting issues may be bypassed to some extent by relying on direct microinjection, at least to the cytosol. Information gathered from single molecule sensing modalities may more accurately reflect the true spaciotemporal distribution of targets and their activity. Moreover, it should allow for assays where two or more targets are monitored in a multiplexed format with the caveat that collecting single molecule FRET data microscopically from intracellular sensors across multiple channels can be quite challenging even with all the tremendous advances in microscope technologies. One approach to addressing this issue is to use different QD colors with a common quencher moiety as previously described to simplify the number of channels required.70 There have also been concerns about how QDs would alter the diffusion dynamics of a target molecule (due to its size/mass) in a single molecule tracking assay, however, this should not be directly relevant here as the information desired is more that of target presence or target activity. QD blinking should also not be a detrimental issue as information would only be collected from the QDs/sensors that are “on.”

Figure 128. (A) Representative NIR fluorescence images of 720QD− peptide−MPA probes in nude mice bearing MDA-MB-231 (λex = 766 nm). (B) The relative fluorescence of 720QD−peptide−MPA probes in nude mice bearing MDA-MB-231 highlighting the change in fluorescence with time. Reproduced from ref 400. Copyright 2014 Elsevier.

Clearly, this is an area that is poised to see dramatic expansion over the near term. Moving forward, however, one critical issue that will need to be clearly understood is that of how stable QD-FRET and related sensor bioconjugates are intracellularly and in vivo. Potential complicating issues include endogenous proteases, nucleases, and the myriad of other biological molecules that make up this environment and that can participate in fouling and corona formation around a given QD bioconjugate. As recently shown, the stability of the organic surface on NPs intracellularly may not be as robust as previously believed and this might require significantly more ligand engineering for specific sensing needs.922 Another

11. PHOTODYNAMIC THERAPY PDT is a tissue-localized oncologic treatment that uses a photosensitizer (PS) or PS agent and illumination with a particular wavelength of light to activate the PS. The PS can be delivered systemically or locally depending upon treatment modality, but is not “activated” until irradiated. In the most

Figure 129. Overview of PDT and potential role of QD-based PDT. Photodynamic processes of PDT and summary of possible mechanisms of PDT initiated by QDs including ROS production directly by the QD, release of toxic ions from QD degradation, and use of the QD as a donor to established photosensitizers. Adapted with permission from MacMillan Publishers Ltd.: Nature Biotechnology932 copyright 2004. 660


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Figure 130. Schematic of biocompatible QDs conjugated to PSs for use in two-photon excitation for PDT. (A) QD coated with a PEG-modified copolymer and coupled to water-soluble porphyrin. Reproduced from ref.941 with permission of The Royal Society of Chemistry. (B) Noncovalent complex consisting of lipid-coated QD embedded with a second generation PS chlorin e6 (Ce6). (C) FLIM image of the noncovalent QDs-Ce6 delivered to HeLa cells highlighting their excited-state lifetime quenching due to FRET. Reproduced from ref 943 with permission of The Royal Society of Chemistry.

Bakalova report, Samia et al. had linked 5 nm CdSe QDs to the silicon phthalocyanine (Pc) PS Pc4 and evaluated the twostep FRET mechanism in a QD-Pc4-oxygen system.359,931 They found a QD-Pc4 EFRET of 77%, but the final 1O2 QY to be only ∼5%, which was significantly lower than the value of ∼40% with Pc4 alone in the presence of molecular oxygen.934 They also observed a fractional amount of triplet energy transfer (TET) between photoexcited QDs and ground-state triplet oxygen (3O2). While this first QD-based system demonstrated a relatively low yield of 1O2, the ability to repetitively stimulate the QDs to promote 1O2 production highlighted the broad potential of these materials as an effective tool in PDT. Generation of 1O2 from QDs directly was demonstrated by Zhou et al. when integrin receptors on pancreatic carcinoma cells were targeted with RGD-coated QDs and exposed to laser illumination.935 However, little discussion or experimental detail is provided as to the mechanism of ROS generation in this system. Subsequent to this study, the generation of ROS directly from QDs was further explored by Yaghini et al.936 Using PEGylated CdSe/ZnS QDs, they reported no direct formation of 1O2 from photoexcited QDs, but when using nonphotolytic visible wavelength excitation, a 1O2 QY of 0.35% was produced. ROS generation was then significantly enhanced via FRET when the QD was coupled to sulfonated-Pcs. Bakalova et al. reported the use of CdSe QD anticluster designate (CD) conjugates in a controlled FRET PDT application.933 The anti-CD90-coated QDs targeted the cell surface of a leukemic cell line. The PSs trifluoperazine (TFPZ) and sulfonated aluminum phthalocyanine (SALPC) were introduced with the QDs on the cell surface and found to significantly decrease cell viability in the presence of UV illumination compared to controls including those against

common mechanism of PDT, energy from the PS excited triplet state is transferred to ground triplet state oxygen creating cytotoxic reactive oxygen species (ROS) such as singlet oxygen (1O2). Since they are extremely reactive, ROS oxidize a range of macromolecules causing irreversible tissue damage but only in the environment in which they are localized and, due to their short lifetimes, only immediately after they are generated. PDT thus provides a targeted tumor treatment and avoids wider ranging systemic effects often associated with traditional chemotherapies. In essence, it is the specific combination of nontoxic individual components (PS, light, and molecular oxygen) that creates a localized cytotoxic effect.929,930 Some persistent challenges to PDT are the generally poor solubility of PSs in biologically relevant media and, as a result, the tendency of these chemicals to aggregate. This aggregation can lead to reduced photochemical activity and poor penetration into targeted tissues and cells.359,931 Several years ago, the concept of using QDs as PSs or PS agents was first suggested and demonstrated.359,932,933 An overview of the PDT process and the various potential roles of QDs within a coupled PDT process is shown in Figure 129. Here, Bakalova et al. highlighted the fact that QDs possess several desirable qualities of a PS or PS agent, including relatively simple and inexpensive synthesis, constant composition, biocompatibility and the ability to target specific tissues, minimal self-aggregation, high photostability, and minimal cytotoxicity in the absence of light but with significant cytotoxic effects under UV irradiation.932 Additionally, the QDs unrivaled multiphoton action cross sections coupled with its effective Stokes shift could allow it to be both an excellent antenna and potent localized sensitizer in tissues at depths far deeper than achievable with current methodologies. Just prior to the 661


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spectrum, making them ideal candidates for deep tissue PDT in the NIR. In TPE mode, FRET efficiencies of over 50% were found as the number of porphyrin molecules per QD was increased to ∼130 and, based on these values, the average donor−acceptor distance was calculated to be 53 Å. Production of 1O2 in the QD-porphyrin complex was significantly higher compared to porphyrin alone, indicating both ET and effective photosensitization under TPE. Li et al. also used TPE with a nonconjugated CdTe QD-copper Pc system.942 EFRET as high as 44% was observed following illumination with a 200 mW, 780 nm femtosecond laser. Higher laser powers led to decreases in efficiency due to increases in the molecular thermal motion and excited-state absorption induced by twophoton absorption. ROS generation was not assessed, though the authors suggest an ET model between the QD and Pc with TPE. More recently, Valanciunaite et al. used PEG-lipid CdSe/ ZnS QDs functionalized with carboxyl, amine or just PEG-only capping ligands to form a noncovalent QD-Ce6 complex and analyzed the FRET performance using both standard fluorescence and TPE, see Figure 130B.943 Complexes were created with QD to Ce6 ratios ranging from 1:0.1 to 1:10. In the carboxyl and nonfunctionalized QD samples displaying a 1:1 ratio of Ce6, a significant increase in the fluorescent intensity of Ce6 was observed compared to Ce6 alone, indicating strong ET and the QD acting as an efficient energy antenna. In fact, QD to Ce6 ratios of 1:5 samples exhibited ∼60% ET efficiency. The center-to-center distance of 38−47 Å between the QD and Ce6 estimated from FRET analysis indicated that the amphiphilic Ce6 molecules were embedded within the PEG-lipid coating at the QD surface, thus enabling high EFRET. Two-photon fluorescent lifetime imaging microscopy (FLIM) of the QD-Ce6 complexes in HeLa cells demonstrated binding, uptake, and high stability inside the cells along with the presence of FRET processes, see Figure 130C. Although direct PDT toxicity assays were not reported, the observed EFRET of ∼50% indicated the complex largely maintained its functionality inside cells and was a facile approach to access QD-based FRET for PDT applications. The use of potentially less toxic Cd-free InP/ZnS QDs in FRET-based PDT was investigated by Charron et al. to better understand the underlying mechanism of 1O2 generation in a FRET-based QD-PS complex.944 Under their preparation conditions, it was estimated that five to 20 Ce6 molecules were covalently coupled via NHS-EDC chemistry to each silicacoated InP/ZnS QD. Using optical spectroscopy, the kinetic rates of the various steps of the complex were systematically examined. It was determined that the rate limiting step of 1O2 generation was ET between the QD and Ce6, meaning that the ET kinetics actually slow the excitation of the PS in this system. A detailed analysis revealed that the rate of 1O2 generation per Ce6 molecule was lower when coupled to a QD, but the fact that multiple Ce6 molecules were coupled to each QD (i.e., larger Ce6 absorption cross section) enhanced the overall 1O2 production. In vitro analysis of the QD-Ce6 complex within the MDA-MB-231 breast cancer cell line revealed a 40% decrease in cell viability compared to negative controls. A similar decrease was observed with molar equivalents of Ce6 alone. This important work helped reveal the underlying mechanistic bottleneck of QD-based PDT and highlighted the need to increase the ET rate between QD and PS by developing strategies to decrease the QD-PS spacing. A different scheme of QD-PS conjugation was proposed by Wu et al. using denatured bovine serum albumin (dBSA) in an

nontargeted normal lymphocytes. While both direct and indirect activation of the PSs may have been observed, the results supported the hypothesis of QD-mediated sensitization of PS to targeted cells. The limitations or precautionary assessment of QDs was also highlighted as Cd ion-induced cytotoxicity was an unwanted potential outcome, as were photoinduced surface chemistries on the QD that may compete with PDT-specific ET pathways and limit 1O2 production.931 The former point could be addressed with a ZnS shell, and the latter addressed by introducing a variety of surface passivating chemistries that also promote biocompatibility. Shi et al. reported 1O2 generation from water-soluble CdTe QDs with the PS TSPP electrostatically bound to the surface.937 They observed a 10-fold enhancement of the extinction coefficient of TSPP at 355 nm when deposited on the QD surface compared to its free form. Further, there was a strong decrease in the overall emission intensity of the QD-TSPP complex, suggesting ET between the two chromophores. Upon illumination with near-UV or visible light, production of 1O2 from the QD-TSPP complex was confirmed with a QY of 43%.937 Marking the progress in developing this systems, this value was significantly higher than the value of 5% reported in the previous CdSe system of Samia,359 though other important considerations such as potential cytotoxic effects from free Cd ions were not addressed. A few years later Ma et al. reported 15% 1O2 QY from SALPCs electrostatically coupled to CdTe QDs.938 Emission spectra of QD, SALPCs, and QD-SALPC composites confirmed the FRET mechanism in the composite. The 1O2 QY values were, however, lower than the value of 36% reported for SALPC alone.939 With a focus on creating an integrated QD-PDT system, Tsay et al. reported coupling two different PSs, Rose Bengal (RB) or Ce6, to a phytochelatin-related peptide and these were then attached to CdSe/CdS/ZnS QDs emitting at 540 or 620 nm, respectively.940 After attempting nonspecific attachment of the PSs, covalent attachment of the PS-peptide segment to the QD surface was carried out via EDC-NHS coupling to form stable complexes. High EFRET of the RB-QD complex was observed compared to the Ce6-QD complex, primarily due to differences in the average FRET donor (QD) to acceptor (RB or Ce6) distance in the two complexes. The latter was compensated for by increasing the ratio of Ce6 displayed on the QD surface. Having confirmed FRET between the QD and PS, 1 O2 production was evaluated and the QY was found to vary between 9% and 31% depending on the PS, acceptor valence, and excitation wavelength. These values were lower than direct stimulation of unconjugated RB or Ce6 with the main limiting factor thought to be inefficient FRET due to either nonoptimal spectral overlap or donor−acceptor distance. Additional detrimental contributions may also have originated from direct excitation of the PS and various nonproductive quenching mechanisms within the conjugate. Despite the inefficiencies, this approach still provided a biocompatible-passivated QD surface, good photostability, and allowed for control over the number of PS conjugates on the QD surface. Given the ultimate goal of accessing deep tissues with these constructs, two-photon excitation (TPE) of QD-PS systems have been examined to gauge their potential. This approach was employed by Qi et al. to generate 1O2 as a result of ET in a covalently bound QD-porphyrin complex, see Figure 130A.941 The biocompatible CdSe QDs had a two-photon absorption cross section ranging from ∼800−2400 Goeppert-Mayer (GM) units corresponding to the 800 to 1100 nm portion of the 662


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Figure 131. (A) Schematic representation of RLuc8-QD complex for BRET-based PDT. (B) Relative growth curves of A549 tumors after various treatments. m-F (0.3 mg m-THPC equivalents/kg); QD-RLuc8 conjugate (10 pmol); coelenterazine (20 μg). *** indicates significant differences compared to the control group (p < 0.0001). (C) Structural characteristics of tumors from different treatment groups highlight the tumor reduction properties of the BRET-based PDT constructs. Reproduced from ref 664. Copyright 2013 Elsevier.

to RLuc8 using EDC coupling. In the presence of coelenterazine, an optimized substrate for RLuc8, bioluminescent energy was shown to transfer to the QD, which in turn had a PL emission centered at 655 nm. The BRET ratio, defined as the ratio of the integrated PL area of RLuc8 to QD, was measured at ∼0.9, reflecting efficient transfer. Compared to controls, the RLuc8-QD complex only showed significant levels of cytotoxicity in A549 cell cultures when in the presence of the PS, Foscan-loaded micelles (m-F), which generated significant levels of ROS. The various components were thought to be predominantly introduced to cells via endocytosis. The effectiveness of this approach was also demonstrated in vitro and in vivo; the former by 50% cytotoxicity and the latter by a 2-fold reduction in tumor volume compared to controls following direct injection of RLuc8-QD conjugates and m-F followed by intravenous injection of coelenterazine, see Figure 131B,C. Both apoptosis and cellular necrosis activity were detected as a result of this treatment. This very promising QDbased approach appears to provide a critical means to avoid external illumination and bypass limitations associated with tissue penetration by light sources. In this context, BRETdriven QD sensitization has also been suggested as a potential treatment for suppressing amyloid-induced oxidative stress in Alzheimer’s disease.946

attempt to improve a range of factors including biocompatibility, PL QY, and access to more efficient FRET rates by decreasing the QD-PS spacing.945 CdTe QDs were first modified with dBSA and EDC-NHS chemistry was then used to link the dBSA-QD to Ce6. Analysis of the conjugate performance revealed an R0 value of 39.6 Å and a QD-Ce6 spacing of ∼43 Å when the number of Ce6 molecules per QD was varied between 10 and 14. Further, FRET efficiencies of ∼90% were observed in the QD-dBSA-Ce6 conjugates and generation of 1O2 was confirmed. The results showed that highly efficient QD-PS FRET could be achieved with the dBSA conjugation scheme, thereby potentially increasing the 1O2 generation rate significantly in a QD-based system. A creative, hybrid approach to PDT with very strong ramifications for future clinical applications was reported by Hsu et al. using Renilla luciferase-immobilized QDs (RLuc8QD) to create a self-illuminating QD system based on BRET excitation.664 By chemically stimulating QD emission with BRET in situ, utilizing the excitation approach demonstrated in the seminal earlier work of Rao,69 the authors demonstrated a way to potentially overcome the limitations of light penetration into deep tissue if the QD complex is effectively targeted to specific disease locations. An overview of the approach is shown in Figure 131A. Carboxylate-coated QDs were covalently linked 663


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Figure 132. Schematic highlighting potential QD-PDT agent pairings in the context of BRET-mediated excitation. Many clinically approved, clinical trial or preclinical trial PS agents are shown along with their optimal spectral window. Only QDs with their facile emission tuning can potentially address the sensitization of all these agents. Reproduced from ref 664. Copyright 2013 Elsevier.

A derivation of PDT known as photochemical internalization (PCI) was demonstrated recently by Yaghini et al.947 In this approach, material sequestered in endosomal or lysosomal vesicles are released intracellularly upon light stimulation. To accomplish this release, CdSe/ZnS QDs functionalized with amine-capped PEG were first covalently coupled using EDC chemistry to the TAT peptide sequence. A negatively charged PS agent, SALPC, was then electrostatically coupled to the TAT-QD complex at various molar ratios with an estimated R0 value of 59 Å for this FRET pair. Steady-state and time-resolved measurements confirmed FRET between the QD and SALPC following excitation of the QD at 405 nm. FRET of the complex in vitro was confirmed following intracellular uptake in human breast carcinoma cells. Extended exposure of the internalized TAT-QD-SALPC complex to radiation at 488 or 635 nm promoted the generation of ROS and intracellular redistribution of the complex due to rupture of the endosomal or lysosomal vesicles. While excitation at 635 nm directly excited SALPC, exposure at 488 nm promoted ET from the QD to SALPC with subsequent vesicle rupture. The authors noted that limited QD redistribution was observed with TATQD controls, inferring the production of ROS was predominantly due to the FRET induced reaction of SALPC with molecular oxygen followed by rupture of the vesicle membrane. Overall, this study demonstrated how cellular uptake of functionalized QDs through endosomal pathways could be redistributed intracellularly by ROS generation as part of the PDT process. Since the concept of FRET-based PDT with QDs was proposed over a decade ago, a significant body of work has been reported to better understand the underlying mechanisms of ROS generation and improve EFRET between QDs and PS. The overall goal is to increase the effectiveness of ROS generation in PDT. While advances have been made in increasing QD biocompatibility and stability, decreasing the QD-PS separation distance for efficient FRET, and investigating TPE to increase PDT tissue penetration, concerns over long-term stability and clearing of QDs containing toxic elements remain. The reader is directed to recent reviews for

an extended discussion of QD and NP-based PDT systems.22,27,948 Nevertheless, due to its cumulative benefits, PDT based on using QDs and especially BRET-driven QD conjugates represents one of the only QD-based materials and processes closest to clinical application. The final implementation will most probably rely on Cd-free QDs to alleviate the heavy metal toxicity concerns. Although, originally described in the context of BRET driven PDT, the schematic shown in Figure 132 highlights why QDs have such strong potential in this role.664 When appropriately configured, only QDs can act as sensitizers for almost any PDT PS agent, with a tunable emission wavelength corresponding to any desired PS agent, in deep tissue with TPE, or even without external illumination when implemented with BRET. Moreover, the efficiency of ROS production can be adjusted or “tuned” upward by modulating the number of PS agents attached per QD. Lastly, the QD-PS conjugates can also accommodate further modifications with a targeting moiety such as a tumor targeting antibody or peptide to help them localize at disease sites in vivo.

12. APPLICATIONS WITH DIAGNOSTIC AND SENSING DEVICES 12.1. Microfluidics

Micro total analysis systems (μTAS) based on microfluidic chips offer many potential analytical advantages, including miniaturization, reduced reagent consumption, faster analysis times, automation, parallelization, and new methods for control of fluid flow, mixing, and delivery of reagents.949−951 The general sensing applications of QDs in conjunction with microfluidics have been reviewed.952 Exploiting FRET for signal transduction in such systems has long been of interest as this can both increase multiplexing capabilities while simultaneously reducing the required optical instrumentation.953−955 As noted already, one of the earliest applications of microfluidics with QDs was capillary flow spFRET assays.68,493−496,516 These examples demonstrated that high-sensitivity detection, low sample consumption, and prevention of photobleaching was 664


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Figure 133. Microfluidic assay with two colors of immobilized QD525 (gQD) and QD605 (rQD) as FRET donors. (A) Scheme of the chemistry showing the in-channel immobilization of QDs, conjugation with probe oligonucleotides, and FRET signaling of target hybridization. (B) The length of FRET-sensitized Cy3 and A647 emission along the length of the channel for three different mixtures of oligonucleotide targets for the gQD-Cy3/ rQD-A647 FRET channels: (i) 49.5/118 fmol, (ii) 49.5/78.0 fmol, (iii) 49.5/39.4 fmol. (C) Correlation between the observed length of FRETsensitized acceptor emission and the amount of target oligonucleotide injected. Curves for the SMN1 target (black square) and uidA target (red circle) displaying the channel length coverage response of each target as a function of the amount of target DNA injected into the channel. (D) Normalized PL spectrum in the absence (dashed line) and presence (solid line) of target. Adapted from ref 958. Copyright 2013 Elsevier.

Figure 134. Microfluidic FRET assay for measurement of the binding kinetics between a QD and acceptor-labeled peptides. (A) Design of the fourarm polyhistidine peptide dendrimer (PHPD) and assay scheme. (B) Microfluidic chip design and numbered observation positions. (C) Photograph of chip. (D) Close up image of the channel. (E) FRET-sensitized Cy5 PL as a function of position along the channel and binding time. (F) Reconstructed progress curve for binding of peptide to QDs. Adapted from ref 283. Copyright 2012 American Chemical Society.

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Figure 135. Compact device for readout of QD FRET assays for proteolytic activity. (A) Design of the device showing a blue electroluminescent (EL) strip, optical filters, compact CCD, and sample chip. (B) PL image of the sample chip with QD-peptide-Cy3 conjugates. The trypsin concentration increases from well a1 to d4. (C) Comparison of results obtained with a fluorescence plate reader and the compact EL device. Adapted from ref 407. Copyright 2009 Elsevier.

possible in a capillary flow format, while another study demonstrated that flow forces in the capillary could improve EFRET through changes in donor−acceptor distance with deformation of biomolecules such as DNA.892 The advantages of microfluidics and the optical sensing capabilities of QDs and FRET are a potentially powerful combination that, surprisingly, has seen only limited research and development to date. One of the first QD FRET assays using the widely popular polydimethylsiloxane (PDMS)-on-glass type of microfluidic chips was reported by Chen et al., who used electrophoretic delivery to immobilize QD-oligonucleotide conjugates on the glass surface of the chip.956 The QDs were conjugated with two oligonucleotide probes: one of which hybridized with capture oligonucleotides immobilized within the channel, and the other of which hybridized with Cy3-labeled target oligonucleotides. The FRET-sensitized Cy3 emission provided a signal for quantitation, while electrophoretic forces could be used to immobilize, strip off, and reimmobilize QDs, and tune hybridization stringency. The assay platform enabled the use of smaller sample volumes and faster assay times than optical fiber assays.956 Tavares et al. also developed a microfluidic platform for FRET-based nucleic acid detection with QD donors.957 In this configuration, SAv-coated QD525 were delivered in-channel by electroosmotic flow and immobilized on the a biotinylated glass surface of a PDMS-on-glass chip. A biotinylated oligonucleotide probe was delivered by electrophoresis and immobilized on the QDs. Cy3-labeled target oligonucleotides hybridized with this probe and, due to fast kinetics within the microfluidic channel, probe sites were saturated immediately. Consequently, the length of the channel over which Cy3 PL was observed was proportional to the amount of target DNA, permitting quantitation without reliance on FRET-sensitized PL intensities or ratios. As shown in Figure 133, a two-color version of this assay was subsequently developed through coimmobilization of SAvcoated QD525 and QD605, and conjugation with two biotinylated probe oligonucleotides that hybridized with Cy3and A647-labeled target oligonucleotides, respectively.958 In another application of microfluidics, Crivat et al. immobilized anionic MAA-coated QDs on a multilayer structure of polyelectrolytes within a PDMS-on-glass microfluidic chip, and conjugated Rh-labeled neurotension peptides to the QDs to enable sensing of TRP activity through the loss of FRET upon hydrolysis of the peptides.959 Long et al. developed a competitive immunoassay for 2,4-D using QD conjugates of 2,4-D-BSA and a Cy5.5-labeled targeting

antibody. Spiked water samples were assayed within minutes using a microfluidic chip with fiber-coupled optics for detection yielding an LOD of 0.5 μM.377 Wang et al. used a microfluidic chip and FRET to study the binding kinetics between QDs and either a four-arm Hisn peptide dendrimer (PHPD) or a His6terminated peptide with subsecond resolution.283 Time correlated with the position of observation along the length of the microfluidic channel, as shown in Figure 134, permitting temporal resolution that would have been difficult with bulk solution measurements of changes in FRET. This type of assay format should be applicable for studying the kinetics of other types of binding events, such as that between a protein-QD conjugate and an acceptor-labeled ligand of that protein. 12.2. Smartphones and Custom Bioanalytical Devices

There is global interest in developing point-of-care (POC) diagnostics that can enable personalized medicine and increase the efficiency and accessibility of health care, particularly in resource-limited settings such as developing countries.560,960 NPs may have important roles to play in this research, as will their integration with consumer electronic devices such as smartphones, and custom devices built around the concept of being mass producible, portable, and low-cost.961 As such, FRET-based sensing configurations with QDs have already been explored in this context. Sapsford et al. developed a compact device for assaying proteolytic activity and inhibition with QD530-peptide-Cy3 conjugates. The device, shown in Figure 135, utilized an electroluminescent strip as an excitation source, a compact CCD camera as a detector, optical filters, and a credit-card sized multiwall chip as the sample holder. The largest dimension of the device was 16 cm. This assay format required a 12-fold smaller sample volume than a microtiter plate assay while still being able to reproduce results obtained with a fluorescence plate reader.407 Similarly, Ho et al. demonstrated hybridization assays with a contact imaging microsystem based on a filterless complementary-metal-oxide-on-semiconductor (CMOS) color photogate (CPG) sensor. Fluidic reservoirs and an optical filter to block blue excitation light from an LED source were placed on top of the CMOS sensor. The fluidic reservoirs contained 10 μL of solution with two colors of QD-probe oligonucleotide conjugate, QD525 and QD625, which were paired with BHQ1and BHQ2-labeled target oligonucleotides. Quenching of QD PL signaled hybridization and the detection limits were 240 nM for the QD525 and 210 nM for the QD625.962 Similarly, Schwartz et al. utilized an active substrate CMOS device in 666


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Figure 136. On-chip hybridization assay utilizing QD FRET with PL lifetime measurements. (A) Assay format showing hybridization of immobilized probe oligonucleotides with dark quencher-labeled targets, and subsequent binding with streptavidin-coated QDs. (B) CMOS microarray device for PL lifetime measurements. (C) Scaling of PL lifetime with target concentration. Adapted from ref 963. Copyright 2008 Elsevier.

modified and rigorously optimized to permit QD FRET assays directly in 100% serum and whole blood with smartphone readout.966 QD625 and A647 were used as the FRET pair, and paper substrates with immobilized QD625 bioconjugates were enclosed in an array of cells in a PDMS chip that limited the optical path length to 250 μm, and a compact platform was developed for smartphone imaging with pulsed illumination from an array of blue LEDs. When the QD625 were conjugated with A647-labeled peptides, it was possible to assay thrombin activity using the recovery of QD625 PL intensity in the R channel of smartphone images. The detection limit was found to be 18 U/mL in whole blood. A competitive binding assay was also demonstrated with a Bt-SAv model system.966 Petryayeva and Algar also demonstrated a homogeneous three-plex assay of proteolytic activity with QD peptide conjugates by pairing QD625, QD525, and QD450 with A647, QSY9, and QSY35 acceptors.397 These QDs aligned with the RGB filters and digital image channels of a smartphone camera. A hand-held, battery-operated UV lamp was used for excitation, and the intensities observed in the RGB channels of digital smartphone images were used for quantitative analysis. This smartphone method, summarized in Figure 138, was able to reproduce quantitative results obtained with a sophisticated fluorescence plate reader, and was able to concurrently track the activity of enterokinase, trypsin, and ChT in single wells across a row of a microtiter plate.397 This study was not only a step toward prospective POC diagnostic applications of QDs and FRET, but also makes QD FRET assays accessible to virtually every research lab. Noor et al. later followed in the footsteps of ref 413, adopting digital image based detection for their aforementioned paper-based QD FRET hybridization assays.562,967 Amplification of FRET signals by drying the paper substrates afforded a detection limit of 450 pmol (150 nM) under illumination using a hand-held UV lamp, albeit 15-fold less favorable than the detection limit for readout with a microscope with a violet laser excitation source and a photomultiplier tube detector.562 As shown in Figure 139, subsequent work combining this assay format with an initial thermophilic helicase-dependent amplification step permitted digital imaging detection of as little as 37 zmol (6.2 fM) of target under UV illumination.967 Use of QDs within smartphones, other customized bioanalytical devices, and similar testing methodologies such as paper strips presents some interesting issues. For simplified

combination with an oligonucleotide microarray for QD FRET assays.963 The device, shown in Figure 136, featured 4096 independent single-photon avalanche photodiode pixel sites and on-chip time-to-digital conversion. Immobilized oligonucleotide probes with a distal Bt linker were hybridized with QSY21 dark quencher-labeled target oligonucleotides, and SAvcoated QD655 were subsequently introduced. The QD lifetime decreased with an increasing concentration of target, and the active substrate format had a lower dependence on probe surface coverage than a conventional microarray format.963 Already ubiquitous throughout much of the world, smartphones continue to grow in their use and availability, and have emerged as promising platforms for POC diagnostics and personalized medicine.961,964,965 These devices offer portability, access to computational power, and both hardwired and wireless connectivity, making them suitable for the acquisition, processing and communication of data and results. Smartphones also have built-in cameras that can be useful for optical measurements. The optical properties of QD are ideal for use with smartphones, other digital imaging devices, and custom-built portable devices because of their excellent brightness, which permits the use of low-power excitation sources, their narrow emission, which can be tuned to match the built-in color filters of consumer camera devices, and their broad absorption spectra, which permit the use of relatively broadband excitation sources without introducing unwanted background in detection channels. Petryayeva and Algar were the first to report the use of digital imaging with consumer devices for readout of FRET-based assays with QD donors.397,413 In an initial study, shown in Figure 137A, QD525 were immobilized as spots on paper substrates that had been modified with thiol ligands, then conjugated with Hisnterminated A555-labeled peptide substrates for the target proteases.413 Under violet light-emitting diode (LED) illumination, digital imaging of an array of spots permitted multiplexed assays of the activity of enterokinase, TRP, and ChT as model protease targets. For quantitative analysis, the digital images were split into red-green-blue (RGB) channels, with QD PL registering primarily in the G channel and A555 PL registering in the R channel. A decrease in the R/G ratio was used to track proteolysis. Analogous results were obtained with a low-cost educational-grade microscope camera, an offthe-shelf webcam, and a smartphone camera.413 In a more recent study, summarized in Figure 137B, this assay format was 667


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Figure 137. (A) Paper-based assays for proteolytic activity with readout via digital imaging under violet LED illumination: (i) Assay scheme showing immobilized QD525-peptide-A555 conjugates and FRET signaling; (ii) spectra for the QD525 and QD525-A555 FRET pair overlaid with the approximate transmission spectra of the built-in red and green camera filters; (iii) digital images of the paper substrates under white light and UV illumination; (iv) example of an assay for trypsin and chymotrypsin showing a time series of three spots of immobilized QD conjugates; (v) comparison between three different digital imaging devices. Adapted from ref 413. Copyright 2013 American Chemical Society. (B) Paper-based smartphone-imaging assays for thrombin activity directly in serum and whole blood: (i) assay scheme showing immobilization of QD625-peptideA647 conjugates and an adjacent reference spot; (ii) photos (left) of the paper-in-PDMS chip under white light and UV illumination, and a photo (right) of the LED illumination platform for smartphone imaging of the chip; (iii) assay data demonstrating detection of thrombin in blood. Adapted from ref 966 with permission of The Royal Society of Chemistry.

fluorescence and especially FRET-based analyses with minimal or simplified detector technologies, no other fluorophore has as much to offer to potentiate this approach. QD chemical and photophysical stability in conjunction with all the previously mentioned FRET properties can make assay implementation relatively simple up to and including enzyme assays in complex matrices such as serum.407,961,966 From a practical standpoint, there are many questions about these prospective technologies that remain to be addressed. For example, whether the cost of synthesizing QDs can be absorbed into a price per unit that is sufficiently low for widespread use in resource poor environments. Although the amount of QD use per assay is miniscule, large scale distribution of QD-enabled technologies could generate concerns about proper disposal of heavy metal waste. This latter point may be addressed by development and

continuous improvement of Cd-free QD materials (Section 3.1), and a full assessment will require consideration of the scope of use and the amount of QDs present in future technologies.

13. LIGHT HARVESTING The idea of coupling natural light harvesting complexes (LHCs) to QDs has been under investigation for several years. A quick glance at the properties of each reveals the potential to develop high efficiency hybrid energy harvesting complexes. QDs offer broad absorption as an antenna and provide strong luminescence, whereas natural LHCs provide high redundancy for accepting and channeling photonic energy. One of the long-term goals of this research is to go even further 668


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Figure 138. Smartphone-based three-plex homogeneous QD FRET assay for proteolytic activity. (A) Assay scheme showing three colors of QDpeptide-acceptor conjugates targeting enterokinase, chymotrypsin, and trypsin. (B) Measurement scheme showing UV illumination of a microtiter plate, digital image capture, and analysis of the image RGB channel intensities. (C) Comparison of data obtained with a fluorescence plate reader and via digital (RGB) imaging. (D) Smartphone image of a column of four samples (i−iv) in microtiter plate, each with a different mixture of the three proteases, as a function of time. (E) The data extracted form image series such as those in panel D can be converted into independent kinetic curves for each of three proteases. Adapted from ref 397. Copyright 2014 American Chemical Society.

Figure 139. Paper-based sandwich hybridization assay with digital imaging readout coupled with helicase-dependent amplification (HDA) of target DNA. (A) Assay scheme showing amplification, the paper-based FRET assay, and digital imaging. (B) Comparison of R/G ratios from digital imaging for increasing concentrations of target of different length. (C) Digital images of samples (1) no sample, (2) negative control sample, and (3) amplified product sample, and column graph showing the quantitative signal contrast. Adapted from ref 967. Copyright 2015 Elsevier.

and integrate QD donors with photosynthetic reaction centers (PRC) in pursuit of understanding and thus recapitulating efficient charge separation. The experimental challenge is significant in bringing together these two seemingly orthogonal biological and inorganic materials to function in a concerted manner as a simple yet efficient entity. An early example of such a hybrid construct was investigated by Li et al., where purple membrane fragments embedded with the light harvesting protein complex bacteriorhodopsin (BR)

were electrostatically self-assembled on the surface of CdTe QDs.968 This bionanosystem was then deposited onto an Au electrode and a stationary photocurrent generated. Analysis of the fluorescent signature of the BR/QD complex revealed severe quenching of the QD emission, suggesting a significant portion of its PL was transferred to BR. The authors suggested a functional model where the 410 nm wavelength light serves two roles. First, it directly excites the QDs, which act as a localized light source to neighboring BR elements. Second, the 669


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Figure 140. Hybrid QD-PRC from Rb. sphaeroides. (A) To-scale interaction of QD and PRC from Rb. sphaeroides showing the active and inactive branches in the electron-transfer cofactor. Positions of absorption/PL maxima for the bacteriochlorophyll (BChl) special pair (P870), BChl monomer (B), bacteriopheophytine (H), and quinone (Q) are indicated for the active branch only. Photons are absorbed by the RC and the QD; an exciton from the QD is transferred to the RC via FRET. Car = carotenoid. (B) Energy-level diagram for the states of the reaction center. QD supplies excitation to the RC by means of FRET. Excitation relaxes to the P870−Qy level. (C) Plot of the molar extinction coefficients of RC and QD570 along with the PL spectrum of QD. (D) Increase of the PL peak at 910 nm associated with the PL emission from the BChl special pair (P870) of RC. Reproduced with permission from ref 972. Copyright 2010 John Wiley and Sons.

main Achilles heel in trying to make this particular type of hybrid QD/PRC assembly a reality. Maskimov et al. also coupled QDs to the LHC phycoerythrin (PE) based on electrostatic interactions, i.e. negatively charged carboxyl groups on the QD surface and some positively charged amino acids on the PE.970 Due to their broad absorption, the QDs were efficiently excited at 266 nm and had a peak emission at 530 nm, which closely matched the absorption of PE. Varying the ratio of QD to PE in solution, the authors found a maximum transfer efficiency of nearly 90%, which corresponds to roughly one QD per two chromophore groups (with each PE protein having ∼30 active chromophoric groups within its macromolecular structure). Schmitt et al. followed up this work by coupling QDs surface-functionalized with either anionic or cationic capping ligands, and examining the ET to the natural LHCs 1 and 2 (LH1 and LH2) and phycobiliproteins, including the previous PE along with phycocyanin and allophycocyanin.971 The ET efficiency for stoichiometric ratios of these QD/protein mixtures was quantified. Five different QDs were used with peak emission ranging from 523 to 640 nm and mixed with the various protein complexes depending

410 nm excitation also affects the BR directly by accelerating an intermediate excited energy state. Therefore, it was the QD/BR combination in conjunction with the 410 nm excitation that provided the stationary photocurrent as opposed to transient photocurrent spikes observed with BR alone. Despite a supposed random orientation of the QDs with respect to BR attachment, this example confirmed the utility of using QDs as nanoscale light antennae to neighboring naturally occurring LHCs. Around the same time, Govorov published an influential treatise describing a hybrid QD/PRC assembly and argued that under realistic conditions, the complex could offer a 100-fold enhancement over the performance of the PRC alone.969 The principle driving factor would be the increased optical absorption cross-section afforded by the QD. This greater cross section, combined with the increased rate of exciton production from the QD could lead to greater excitation of the PRC for subsequent charge separation. Careful design, namely through nanoscale orientational control of the components with respect to one another, was suggested to play a critical role. Indeed, with the benefit of hindsight, uncontrolled biomolecular orientation would repeatedly prove to be the 670


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Figure 141. Purple membrane-QD hybrid. Photons are absorbed by the QD immobilized on the surface of the PM containing BR. (A) Each BR contains one retinal chromophore (red) located in the center of the PM, ∼2.5 nm from each membrane interface. An exciton from the QD is transferred via FRET initiating retinal photoisomerization and eventual proton pumping through the PM. (B) Proteoliposomes membrane fragment containing BR (blue) with immobilized QDs. Illumination with orange light directly excites BR. Higher energy blue photons directly excite QD, which then engage in FRET with retinal (shown in red.). Absorption of photons by BR induces proton pumping inside proteoliposome with photoinduced increase of pH in surrounding solution. Objects are to scale. Adapted from ref 973. Copyright 2010 American Chemical Society.

Figure 142. FRET of QD-β-PE complex. (A) Model of QD-β-PE assembly with estimated donor−acceptor separation distances. (B) Donor quenching and acceptor sensitization as a function of the ratio of β-PE to QD. Adapted from ref 302. Copyright 2009 American Chemical Society.

on the spectral overlap. Quenching of the QD fluorescence ranged from 70 to 80% when coupled to PE, 50−65% when coupled to phycocyanin, and 45−70% when coupled to allophycocyanin, with only slight quenching observed between the QDs and LH1 or LH2 complexes (though ET in the two latter hybrid structures was not discernible). The results revealed that there was not a straightforward observable correlation between QD donor quenching and protein complex acceptor reemission or between the ET and the spectral overlap integral, J, in this case. The reasons for this could not be determined from the experimental setup, partially due to the fact that the electrostatically driven QD-protein structures offered little control over the orientation of the two components relative to one another. Despite the detection of ET in certain hybrid structures, this work highlighted the significant challenge of asserting nanoscale control over the orientation of acceptor and donor moieties in these types of constructs. Electrostatic assembly was also employed by Nabiev et al. in a seminal study that created a hybrid complex composed of CdTe QDs and PRC isolated from the purple bacteria Rhodobacter sphaeroides.972 The same basic principle of using the QD, with its large optical cross-section and optimized spectral emission, as the primary antenna for the PRC was

explored by taking into consideration the various active subcomponents of the PRC responsible for the energy absorption and eT steps, see Figure 140A. An energy diagram of the hybrid complex was suggested with the QD supplying energy to the PRC via FRET (Figure 140B), and this was subsequently confirmed as the primary mechanism based on QD quenching and fluorescence lifetime analysis. Based on the underlying spectral overlap (Figure 140C), optical enhancement of the complex compared to PRC alone (Figure 140D) was confirmed to occur at wavelengths where the QD strongly absorbed. This work was significant in that it was the first demonstration of coupling the natural light harvesting PRC to QDs, albeit with no control over the orientation of the two components with one another. Around the same time, Rakovich et al. reported on the performance of a “nanoconvertor” consisting of water-soluble CdSe/ZnS or CdTe QDs coupled to BR in purple membrane from Halobacterium salinarum (Figure 141).973 As implemented, the QD was immobilized on the surface of the purple membrane containing BR and served to harvest light and transfer an exciton via FRET to retinal, the chromophore within BR, initiating the photocycle and proton pumping through the purple membrane. FRET within the QD-BR complex was confirmed by both steady-state PL and time671


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Combining natural LHCs and QDs appears to offer significant potential for understanding and improving light harvesting efficiency for a range of applications spanning the fields of biology, optics, and electronics. While control has been exerted over the spectral overlap of the QD and LHC, one of the challenge moving forward continues to be control over the nanoscale spatial organization between the components. Instances of QD immobilized to BR-containing purple membrane have been the clearest demonstration of such spatial control,973,974 though development of other oriented hybrid architectures are needed to facilitate their utility in a range of nanotechnologies. This orientational control will require significant improvements to QD surface chemistries and perhaps even the LHC structures as well to optimize their bioconjugation in a manner that allows the two materials to be melded together in a concerted and predetermined manner so as to allow more efficient interactions. Even if this control were achievable, the limited long-term stability of complex LHC proteins would still be a continuing issue. From a pure research perspective, we often forget that plants invest considerable resources in replacing proteins continuously which is not something easily accomplished in research formats. Although the focus of this Section is around LHC and similarly functional proteins, there are also other related structures that can contribute here in the context of QDs and FRET. These include the QD-based DNA photonic wire assemblies along with the BRET driven constructs described in Section 5. In this context, these composite QD biomaterials could directly contribute as part of the antennae or light focusing/directing sections. Indeed, it is not hard to envision self-assembly driven supramacromolecular structures where multiple QD-based DNA photonic wires are arrayed around and connected to a central reaction center. Clearly, research in this area will provide an important opportunity for learning how to create next generation high efficiency biomimetic hybrid light harvesting and charge separation devices.

resolved PL studies of QD donor quenching and a decrease in the average lifetime of the QD PL, respectively. A significant increase in efficiency of BR activity was observed upon QD coupling by monitoring pH changes in proteoliposome suspensions containing the purple membrane complex in the presence or absence of immobilized QDs. The increase was largely due to the QD harvesting light energy outside of the absorption spectrum of the BR complex and exciton transfer into the proton-pump pathway. Another significant contributing factor was the coupling of QD to BR, which was within the purple membrane and which offered a level of control over the spatial orientation of the active elements of this complex. A follow up study by the same group reported a significant enhancement of 20- and 40-fold in the nonlinear refractive index (n2) of the hybrid QD-BR complex compared to the n2 of either the BR-containing purple membrane suspension or QD, respectively.974 Through examining a series of controls, a strong correlation was observed between n2 enhancement and high efficiency FRET, which suggests the origin of the enhancement lies in the photoactive retinal molecule in purple membrane. However, questions remain to develop a complete understanding of the other contributing factors involved in n2 enhancement in this complex. The light harvesting complex β-phycoerythrin (β-PE) was one of three fluorescent protein structures coupled to the surface of QDs by Medintz et al. following its modification with SAv to allow binding to the biotinylated surface of QDs, see schematic in Figure 142A.302 The QD spectral emission was tuned to overlap with the primary absorption peak of β-PE, thus optimizing the spectral conditions for FRET. Evidence of FRET was observed by significant QD quenching and concomitant β-PE sensitization as the ratio of protein per QD was increased (Figure 142B). Analysis of the experimental EFRET in conjunction with detailed modeling to account for the heterogeneity of this structure revealed a FRET efficiency of 1 × 106 Da of protein were carried per QD into cells with this approach. A similar SAv-Bt approach was also used by Griep et al.,975 where it was shown that activation of a BR-based electrode was enhanced by coupling BR to CdSe/ZnS QDs. Optimization of the QD emission with BR absorption provided a 23% amplification of the photovoltaic response compared to BR alone under white light illumination, and both radiative and nonradiative ET were implicated. These results further confirmed the effectiveness of developing a hybrid complex with control over both the spectral overlap and spatial distribution between the two components.

14. RELATED AND MISCELLANEOUS STRUCTURES/PROCESSES This Section provides an overview of three QD eT configurations that do not fall naturally into the previous headings. The first looks at utilizing QDs with electrodes, where the QD acts as an intermediary and relays electrons to or from the electrode within some biological assembly or biosensing process(es); this transfer can be under photoexcitation conditions as well. The second provides an overview of utilizing QD photoexcitation to generate electrons that are then transferred to enzymes or cofactors as part of biological redox chemistry. Lastly, an overview of H2 production is provided. Although clearly far less “bio” oriented, the latter process still utilizes QD ET and takes place in an aqueous environment making this important research area warrant some overview. Similar to many of the previous Sections, the examples highlighted are meant to be representative of the goals and experimental approaches being utilized to provide the reader with an appreciation of what is being undertaken and why. 14.1. Electrode Systems Incorporating Quantum Dots

The quantum effects of QDs clearly provide remarkable electronic properties to many developing applications. For example, the integration of QDs into solar panels continues to be a tremendously active area of investigation, see for example 672


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higher localized density which serves to increase the probability and rate of eT. Tables 4 and 5 provide a summary of several

refs 976 and 977. Notably, many of the same QD electronic properties can contribute beneficially to sensing and other applications or active configurations as part of their interactions with electrodes. When dealing with electronic systems, particularly electrodes, it is surface effects that oftentimes dominate the function, and here the nontrivial QD surface area can contribute extensively. Beneficial properties include an increase in electron relaying through tunneling, which serves to improve the sensitivity of electrical bio/chemical sensors. The QDs also present a large surface area-to-volume ratio, making them very sensitive to any analytes in their environment. On an electronic level, QDs will maintain a linear response to applied potentials, and generally demonstrate single electron tunneling, thus avoiding potential Coulomb blockades that could interfere with the currents.976,978 When functionally incorporated onto an electrode, the QD will accept electrons into the valence band (either from the electrode or the analyte) and transfer the electrons from its conduction band, again to either the electrode or the solution depending on the current direction (Figure 143).978 In many cases, QDs can also augment the level of current being monitored with simultaneous photoexcitation (vida infra).

Table 4. Overview of Glucose and Peroxide Sensors Utilizing QD-Modified Electrodes QD

co-component nanostructures

electrode

LOD [nM]

integration of glucose oxidase for glucose detection ITO CdS. They also varied the size of the QDs and compared them to the corresponding micropowders, which were unable to photocatalyze NADH regeneration. It was observed that the smaller the utilized QD the more efficient the regeneration, and this result was attributed to not only a greater surface area-tovolume ratio, but also to the relative increase of the CT rate in comparison to that of the electron−hole recombination.1027

Subsequent reports from the same group exploited similar systems but added other SiO2 or TiO2 support systems that increased the efficiency1028,1029 or provided other benefits such as the automation of the reaction within a microfluidic cell.1030 14.3. Hydrogen Production

The proposed hydrogen economy where H2 forms the basis of a clean medium for energy storage and transportation power has garnered much research interest.1031,1032 Though this possibility is still somewhat far off, initial steps into how to obtain and store H2 are underway. For this goal, QDs may play a role in photocatalyzing the splitting of water to obtain the desired H2.1033 Water splitting under photoirradiation was first demonstrated with large band gap (TiO2) electrodes in 1972 by Fujishima and Honda,1034 and since that time the integration of small band gap NPs such as QDs as a means to increase efficiency has continued to push the field. QDs present various characteristics that make them of interest in splitting water. These are primarily their large extinction coefficients in the visible light range of the electromagnetic spectra1035 along with their capability to quickly and efficiently undergo chargeseparation under illumination.1036 If the subsequent transfer of the excited electron is faster than the charge recombination rate, then the QD can efficiently catalyze the desired reactions similar to that seen in the previous enzyme examples. As the reactions take place primarily in water, they somewhat meet the criteria of a biocompatible application, and given the strong interest in this area, coupled to the QD ET focus here, some discussion is warranted. The field is vast and almost all aspects of the systems under study are continuously being optimized with the aim of improving overall performance. Specifically focusing on this improvement perspective allows us to provide an overview of some of the research areas related to the current topic. To begin with, the chemistry of the QDs themselves needs to be optimized. Since charge separation within the QDs optimizes the photocatalytic capacity, then mechanisms that normally improve QY should also apply and contribute to H 2 production.1037 As such, multiple groups have looked at the difference between core-only QDs as compared to core/shell QDs. As expected, core/shell QDs present increased overall efficiency both in the rate of H2 production as well as in stability.1036,1037 If the overall absorbance of the QD is maintained as a constant, then a thicker shell (CdS) appears preferable to a larger core (CdSe).1036 One example of an “outside the box” core/shell structure substituted a thin layer of 676


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Figure 148. (A) Schematic of QDs with three types of surface ligands. (B) H2 evolution as a function of time under visible light irradiation for the varying ligand capped QDs along with a Pt cocatalyst. (C) Schematic of energy levels for H2 generation demonstrating the disadvantage of surface trap states. Reproduced from ref 1039 with permission of The Royal Society of Chemistry.

Figure 149. Schematic of CdS-TiO2 nanotubes charge transfer and energy levels. Reproduced from ref 1044. Copyright 2015 Elsevier.

α-TiO2 as the shell on CdSe QDs (Figure 147).1038 The inclusion of the α-TiO2 creates a band offset in the conduction band of the CdSe, where the electron can transfer to the shell improving charge separation and resulting in an increase in H2 production from 89 to 240 μmol g−1h−1. Surface ligands also play a similar role in optimizing eT and are crucial for minimizing QD surface defects. To this end a comparison between the H2 photogeneration obtained with CdSe/CdS QDs coated with either PAA, 3-mercaptopropionic acid (3MPA), and 2,3-dimercaptosuccinic acid (DMSA) was undertaken by Wang et al.1039 They demonstrated that photogeneration correlated with QD PL in the order DMSA > 3MPA > PAA (see Figure 148). A further variable is the overall shape of the NPs and, in a study comparing CdS nanorods to spheres functioning with a Pt cocatalyst, it was observed that

the rods increased H2 production nearly 50-fold to 223 μmol g−1 h−1.1040 As mentioned, the first and still one of the most common materials for H2 production is TiO2. The large band gap of TiO2 limits its range of absorption of natural sunlight, and to make up for this deficiency, QDs can be integrated within TiO2 to enhance visible light absorption (Figure 149). For example CoSx QDs have been integrated into TiO2 spheres increasing H2 production 35-fold to 838 μmol g−1 h−1.1041 Similarly QDs can be integrated into TiO2 films,1042,1043 nanorod and nanotube arrays,1044,1045 and molecular sieves.1046 A slightly different approach utilized hematite as the photoelectrode since it is a porous material with a medium band gap between QDs and TiO2, which can potentially improve charge transport in the system and increase overall H2 production.1047 Another option that can increase charge separation is the use of carbon 677


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Figure 150. (A) Cobalt structures (1, 2) used as cocatalysts in hydrogen production. (B) Electron level diagrams of QD in combination with cobalt structure 1. H2 generation as a function of time with visible light irradiation comparing the QD with QD-cobalt structure 1 (C) or 2 (D) complex. Reproduced from ref 1057 with permission of The Royal Society of Chemistry.

complexes.1053 Cobalt is cheaper than Pt and Pd, and the complexes can self-assemble to the QDs along with accepting the electrons produced on the picosecond time scale, which is faster than the typical charge recombination time (See Figure 150).1033,1057,1058 The cobalt complexes are then capable of transferring the electrons to protons obtaining the desired product.1058 Another important aspect of H2 production is in tailoring the system to the medium in which they are to be utilized. The most common conditions are slightly acidic aqueous solutions containing Na2S and Na2SO3 as sacrificial reagents.1054,1059 Formic acid can provide another alternative and has been postulated as a relatively easy form of storing hydrogen in a chemical manner that can then be converted into H2 on demand.1037 This conversion has been achieved at room temperature by using CdS QDs along with a cobalt cocatalyst without the need of a precious metal with an observed capability of producing 1.16 × 105 μmol g−1h−1 H2 while demonstrating long-term structural stability.1060 A further variable available in designing a QD-based H2 production cell is the QD alignment. Using a quaternary ammonium copolymer to stabilize and create clusters of snake-like CdS QDs, the Bicak Group was able to demonstrate stabilization of the QDs in aqueous solution. H2 production was slightly varied by selecting different proportions of the block polymers used to stabilize the QDs.1061 For many of these photoelectron transfer systems, including the previous enzyme examples, the limiting step is hole-scavenging (generally accomplished with triethanolamine or ascorbic acid). Beyond the possibility of chemically designing a more efficient hole scavenger, tailoring of the respective QDs may allow for increased hole removal and ultimately better translational technologies. Although somewhat unrelated in the aggregate, each of these approaches obviously comes with their own set of benefits and liabilities and these will also factor into how they evolve and are

nanostructures, where they serve as acceptors of electrons (or holes) generated in the QD upon irradiation. Multiple examples can be found of QDs either grown or deposited onto graphene/ graphene oxide/graphitic carbon nitride sheets.1048−1050 Of particular interest is the use of GO in combination with heterostructured ZnO/PbS QDs to obtain multiexciton generation.1051 In this configuration, high energy photons create a charge separation but the additional energy is not dissipated in heat as normal, but rather in creating another electron−hole pair, which can result in over 100% QYs. The GO is able to absorb the charges at a sufficient speed so as to allow the naturally occurring multiple exciton properties of the QDs to increase the photocatalytic efficiency by 3-fold. Other materials that can quickly absorb charges include NiO electrodes, which are particularly adept at absorbing holes created during the reaction.1052,1053 H2 production generally requires the use of precious metals such as Pt and Pd to reduce the reaction’s overpotential in water, which is often the case when using QD integratedsystems as well. One study showed a 0.2% efficiency when CdS was present alone but an increase to 40% and 53% with Pd and Pt, respectively.1054 Other catalysts that do not work by the same mechanism of overpotential reduction have been developed by taking advantage of the specific capability of QDs to conjugate to synergistic moieties, or, alternatively due to size constraints, be placed around the synergistic moieties.308 In one representative example, AuNPs (14 nm diameter) were decorated with multiple 3−5 nm CdS QDs increasing the H2 output as compared to the QDs alone through the gold’s capability to act as a hole scavenger.1055 A more economic option to Pt or Pd is the use of simple nickel ions complexed to the CdS/CdSe QDs.1056 Though not as efficient as Pt or Pd, these nickel ions (if found with sulfur) could work as a sensitizer increasing proton reduction. Possibly the largest group of cocatalyst along with QDs is the use of cobalt-based 678


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types of enzymatic activity can soon be expected. The latter processes are especially exciting as they can provide for far more sensitive (and potentially specific) high-throughput pharmaceutical screening assays. As an interesting aside, an in-depth assay where a QD biosensor is applied to screening the interactions of thousands of different molecules in a compound library with an enzyme or receptor protein, for example, has yet to be reported in the literature. Hopefully, such a study will come to fruition soon. The nearer to longer term will also see sensors with QD donors make a concerted migration to cellular and possibly even in vivo applications. For the cellular work, the primary impediment remains finding a robust method to deliver the sensor to the cellular cytosol or other targeted subcellular organelle, while for the in vivo utility, finding NIR dye acceptors that emit near or within the tissue transparency window with effective brightness remains problematic. Any in vivo application will also have to deal with potential effects from protein corona formation on the QD, including how the activity of the “sensing” protein or nucleic acid components within these assemblies are altered by the complex constituents of this environment.50,51,53,154 Additionally, whenever the possibility of in vivo application of QDs is discussed, the unresolved issue of QD toxicity soon joins the discussion. As mentioned at the beginning of this review, acute or overt QD toxicity has not been seen in some preliminary mammalian studies,124−126 however, far more information from strictly structured and parametric studies are still needed. Moreover, the amount of information available on cellular studies of QD toxicity is growing rapidly and the methods for analyzing this large data set are also becoming increasingly sophisticated. Indeed, a meta-analysis of the largest data set collected to date for cellular toxicity of Cd-containing QDs was recently reported.1067 This study drew from 307 individual publications to assembly 1,741 cell viability-related data points (including 514 IC50 values) each of which is associated with 24 qualitative and quantitative attributes describing the experimental conditions and material properties. Using random forest regression models, it was shown that QD cytotoxicity was close correlated to QD surface properties such as the presence of a shell, diameter, ligand type and further surface (bio)modifications.1067 It is also important to note that the role of QDs for in vivo research would most likely be one of a wellequipped and versatile tool to perform targeted biosensing or track drug delivery, and that, in the context of an actual NPbased therapeutic, the final material will most likely not be a Cd-containing QD.44 It is quite probable that the near term will also witness development of QD FRET constructs specifically intended for super-resolution and similar imaging applications. These constructs may be based on some pcFRET configuration given its excellent ability to dramatically switch construct PL emission from one wavelength to another across a significant portion of the spectrum.264,614 The issue in utilizing these constructs experimentally will now be that of stealthily infiltrating them into a cellular biological process or cellular location such that they provide useful information back to the experimentalist without perturbing that activity. This task will obviously be predicated on a careful bioconjugation strategy. Within the role of a FRET acceptor in a (bio)configuration, QD utility is still vastly underutilized and underexploited. Almost all of the examples reported to date rely on BRET/ CRET excitation or, alternatively, the use of long-lifetime metal chelate donors. This dependence on special donors arises due

applied in the future. The more interesting point to be made here is that they also represent ways in which QDs and ET in a biological context can be exploited, but in a manner that is quite different from the rest of the previous discussion. This also bodes well for the future as it suggests that other “nontraditional” applications for this materials/process combination can also be expected as a need for them is developed.

15. CONCLUSIONS AND OUTLOOK A cumulative examination of the work reviewed here allows us to put forth some conclusions about the current state of this research field and some of the many potential applications that will soon emerge from it. In the role of a FRET donor, QD utility has reached the point where it can be used in a facile and predictable manner. As amply confirmed by the myriad of examples cited herein, calculating the spectral overlap function and R0, experimentally determining FRET efficiency, and estimating donor−acceptor separation distance along with the effect of multiple acceptors per QD donor are all now relatively well understood, accepted and routinely applied. The one persistent, remaining issue is that of explicitly defining the location of the QD’s dipole and its orientation, especially given the nontrivial QD size compared to molecular fluorophores.1062 The QD exciton wave function is typically assumed to have its highest magnitude at the center of the QD (core),100,351,1063−1066 and, exempting materials with known recombination events in the shell, defining this center as the donor “location” or origin for FRET has become the common working assumption.28−30,72 In terms of donor−acceptor dipole orientation, it is important to consider that almost all QDacceptor (bio)configurations are randomly assembled with the location of the acceptor(s) and their dipole relative to the QD core being quite heterogeneous across an ensemble even if they are all placed centrosymmetrically.28−30,72,263 This heterogeneity arises from the inherent nature of the bioconjugation chemistry and is clearly exacerbated when multiple acceptors are displayed around a QD. Additionally, it is almost impossible to know or fix the dipole orientations. Suffice to say that, from a functional perspective, assuming a random dipole orientation (κ2 ≈ 2/3) appears to be viable and has not proven to be a significant impediment to using QD donors for FRET, especially in (bio)sensing applications. Moreover, most donor−acceptor distance values extracted from such ensemble constructs generally appear reasonable with the known sizes of the participants, and how they would assemble to each other. In other words, assumed dipole orientations should not make much of a difference for most generalized QD donor-FRET bioapplications unless an absolute measurement with subÅngström precision is needed or specific types of single molecule studies are being undertaken. Aside from the latter, this issue is, to some extent, considered more academic for ensemble applications with the realization that no definitive answer is yet available. Looking at the heavy application of QD donors in all manner of biosensing formats suggests that much more of the same can be expected in the near term. This includes applications such as proteolytic monitoring along with other targeted enzyme sensing, small molecule sensors, and molecular beacons. As witnessed by the concentric FRET sensors (Section 5.2.5.2), the sophistication and capabilities of these sensors will most certainly increase. Moving beyond the typical “proof of concept” formats utilizing biotin−avidin interactions or a thrombin aptamer, sensors targeting new molecules and new 679


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15 years have probably had a profound influence to some degree on many other applications where QD FRET is important, such as solar energy harvesting and conversion along with related types of optics and displays. Even if only limited to the confines of a “biological” configuration as we have defined it here, it is still quite appropriate to say that the future of QDs and energy transfer is getting brighter and still growing.

to the photophysical constraints imposed by the cumulative effects of the QD’s broad and continuously increasing, blueshifted absorption profile, coupled to its longer lifetime relative to organic dyes and fluorescent proteins (Section 5.3.1). However, the ability to dramatically reduce background fluorescence and scattering, along with engaging in multistep FRET processes, does suggest excellent potential for cellular/ tissue biosensing and imaging, which is an area almost wholly unexplored with these materials. For multistep ET in particular, the QD appears to function as an effective central energy harvesting scaffold and relay for coupling BRET or long-lifetime donors to multiple downstream acceptors.286,293,668 Indeed, such utility within QD/DNA-based photonic wire configurations seems to hold a lot of promise for nanoscale energy harvesting, molecular electronics and alternative computing, along with possible cryptography applications. QD ET to AuNPs and other metal NPs along with eT bioapplications are also vastly underutilized and underexploited. Here, the primary impediments arise from the lack of a full understanding of the properties of participating materials, such as AuNPs, or the effects of an aqueous environment along with biological interfacing on QD eT. Using the example of AuNP acceptors, QD ET interactions with these materials vary significantly with the AuNP size but not in a manner that lends itself to a clear and systematic analysis. For smaller AuNP acceptors (

Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A

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