Sebastián A. Díaz

Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications

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.

Photoswitchable water-soluble quantum dots: pcFRET based on amphiphilic photochromic polymer coating.

A novel surface architecture was developed to generate biocompatible and stable photoswitchable quantum dots (psQDs). Photochromic diheteroarylethenes, which undergo thermally stable photoconversions between two forms with different spectral properties in organic solvents, were covalently linked to an amphiphilic polymer that self-assembles with the lipophilic chains surrounding commercial hydrophobic core-shell CdSe/ZnS QDs. This strategy creates a small (∼7 nm diameter) nanoparticle (NP) that is soluble in aqueous medium. The NP retains and even enhances the desirable properties of the original QD (broad excitation, narrow emission, photostability), but the brightness of its emission can be tailored by light. The modulation of emission monitored by steady-state and time-resolved fluorescence was 35-40%. The psQDs exhibit unprecedented photostability and fatigue resistance over at least 16 cycles of photoconversion.

Modulation of a photoswitchable dual-color quantum dot containing a photochromic FRET acceptor and an internal standard.

Photoswitchable semiconductor nanoparticles, quantum dots (QDs), couple the advantages of conventional QDs with the ability to reversibly modulate the QD emission, thereby improving signal detection by rejection of background signals. Using a simple coating methodology with polymers incorporating a diheteroarylethene photochromic FRET acceptor as well as a spectrally distinct organic fluorophore, photoswitchable QDs were prepared that are small, biocompatible, and feature ratiometric dual emission. With programmed irradiation, the fluorescence intensity ratio can be modified by up to ∼100%.

Quantum dots as templates for self-assembly of photoswitchable polymers: small, dual-color nanoparticles capable of facile photomodulation

A photomodulatable amphiphilic polymer has been synthesized with a backbone of poly[isobutylene-alt-maleic anhydride] and pendant dodecyl alkyl chains, Lucifer Yellow (LY) fluorescent probes, and diheteroarylethenes photochromic (PC) groups. The latter serve as reversible UV-activated FRET acceptors for the LY donors. We characterized the spectral and switching properties of the polymer in an organic solvent (CHCl(3)). In an aqueous medium the polymer forms polymersomes, constituting fluorescence probes ~75 nm in diameter. Self-assembly of the polymer on the surface of a quantum dot (QD) serving as a template creates a dual-color photoswitchable nanoparticle (psNP) with improved properties due to the increase in polymer density and efficiency of PC photoconversion. The psNP exhibits a second QD red emission band that functions as an internal standard requiring only a single excitation wavelength, and is much reduced in size (<20 nm diameter) compared to the polymersomes. The QD template also greatly increases the depth of modulation by photochromic FRET of the LY emission monitored by both steady-state and time-resolved (lifetime) fluorescence (from 20%→70%, and from 12%→55%, respectively).

Photoswitchable semiconductor nanocrystals with self-regulating photochromic Förster resonance energy transfer acceptors

Photoswitchable molecules and nanoparticles constitute superior biosensors for a wide range of industrial, research and biomedical applications. Rendered reversible by spontaneous or deterministic means, such probes facilitate many of the techniques in fluorescence microscopy that surpass the optical resolution dictated by diffraction. Here we have devised a family of photoswitchable quantum dots (psQDs) in which the semiconductor core functions as a fluorescence donor in Förster resonance energy transfer (FRET), and multiple photochromic diheteroarylethene groups function as acceptors upon activation by ultraviolet light. The QDs were coated with a polymer bearing photochromic groups attached via linkers of different length. Despite the resulting nominal differences in donor-acceptor separation and anticipated FRET efficiencies, the maximum quenching of all psQD preparations was 38±2%. This result was attributable to the large ultraviolet absorption cross-section of the QDs, leading to preferential cycloreversion of photochromic groups situated closer to the nanoparticle surface and/or with a more favourable orientation.

Evaluating Dye-Labeled DNA Dendrimers for Potential Applications in Molecular Biosensing

DNA nanostructures provide a reliable and predictable scaffold for precisely positioning fluorescent dyes to form energy transfer cascades. Furthermore, these structures and their attendant dye networks can be dynamically manipulated by biochemical inputs, with the changes reflected in the spectral response. However, the complexity of DNA structures that have undergone such types of manipulation for direct biosensing applications is quite limited. Here, we investigate four different modification strategies to effect such dynamic manipulations using a DNA dendrimer scaffold as a testbed, and with applications to biosensing in mind. The dendrimer has a 2:1 branching ratio that organizes the dyes into a FRET-based antenna in which excitonic energy generated on multiple initial Cy3 dyes displayed at the periphery is then transferred inward through Cy3.5 and/or Cy5 relay dyes to a Cy5.5 final acceptor at the focus. Advantages of this design included good transfer efficiency, large spectral separation between the initial donor and final acceptor emissions for signal transduction, and an inherent tolerance to defects. Of the approaches to structural rearrangement, the first two mechanisms we consider employed either toehold-mediated strand displacement or strand replacement and their impact was mainly via direct transfer efficiency, while the other two were more global in their effect using either a belting mechanism or an 8-arm star nanostructure to compress the nanostructure and thereby modulate its spectral response through an enhancement in parallelism. The performance of these mechanisms, their ability to reset, and how they might be utilized in biosensing applications are discussed.

Understanding energy transfer with luminescent gold nanoclusters: a promising new transduction modality for biorelated applications

Ultrasmall noble metal and especially gold nanoclusters (AuNCs, ≤2 nm diameter in size) display a range of unique quantum confined and photophysical properties which are far different from their larger-sized nanoparticle counterparts or that of the bulk parent material. Amongst these properties, the photoluminescence of gold AuNCs has stimulated much interest for biological applications due to a combination of their small size, high photostability and range of emissions depending on size and surface stabilizing ligands, including near-IR emission. The dearth of robust Förster resonance energy transfer (FRET) donors and acceptors available to populate the latter spectral range is also driving strong interest in applying AuNCs for similar utility and especially for biosensing. However, the exact mechanism of how AuNCs engage in this type of energy transfer (ET) is still not defined and accumulating evidence indicates that it is not by a classical Förster process although, interestingly, many of the same characteristics and photophysical requirements seem to be present and even many-times required. Here, we summarize the state of the art in AuNC ET studies with a special emphasis on relevance to biological utility ranging from diagnostics to distance measurements along with describing the different ET mechanisms that have been ascribed with their use. Due to its corresponding importance in this discussion, we provide a brief overview of how these materials are synthesized, the current understanding of how their photoluminescence originates, and some related information on silver nanocluster (AgNC) ET along with related processes such as chemically-induced ET. A perspective and outlook on how this area will develop in the future is also provided.

Bridging Lanthanide to Quantum Dot Energy Transfer with a Short-Lifetime Organic Dye

Semiconductor nanocrystals or quantum dots (QDs) should act as excellent Förster resonance energy transfer (FRET) acceptors due to their large absorption cross section, tunable emission, and high quantum yields. Engaging this type of FRET can be complicated due to direct excitation of the QD acceptor along with its longer excited-state lifetime. Many cases of QDs acting as energy transfer acceptors are within time-gated FRET from long-lifetime lanthanides, which allow the QDs to decay before observing FRET. Efficient QD sensitization requires the lanthanide to be in close proximity to the QD. To overcome the lifetime mismatch issues and limited transfer range, we utilized a Cy3 dye to bridge the energy transfer from an extremely long lived terbium emitter to the QD. We demonstrated that short-lifetime dyes can be used as energy transfer relays between extended lifetime components and in this way increased the distance of terbium-QD FRET to ∼14 nm.

3,4-Dihydroxyphenylalanine Peptides as Nonperturbative Quantum Dot Sensors of Aminopeptidase

Fluorescence-based assays for hydrolases that cleave within the substrate (endopeptidases) are common, while developing substrates for proteases that selectively cleave from peptide termini (exopeptidases) is more challenging, since the termini are specifically recognized by the enzyme and cannot be modified to facilitate a Förster resonance energy transfer (FRET)-based approach. The development of a robust system that enables the quenching of fluorescent particles by simple amino acid side chains would find broad utility for peptide sensors and would be advantageous for exopeptidases. Here we describe a quantum dot (QD)-based electron transfer (ET) sensor that is able to allow direct, quantitative monitoring of both exopeptidase and endopeptidase activity. The incorporation of 3,4-dihydroxyphenylalanine (DOPA) into the sequence of a peptide allows for the quenching of QD photoluminescence through an ET mechanism. DOPA is a nonproteinogenic amino acid that can replace a phenylalanine or tyrosine residue in a peptide sequence without severely altering structural properties, allowing for its introduction at multiple positions within a biologically active peptide substrate. Consequently, the quenching system presented here is ideally suited for incorporation into diverse peptide substrates for enzyme recognition, digestion, and activity sensing. Our findings suggest a broad utility of a small ET-capable amino acid side chain in detecting enzyme activity through ET-mediated QD luminescence quenching.

Elucidating Surface Ligand-Dependent Kinetic Enhancement of Proteolytic Activity at Surface-Modified Quantum Dots

Combining biomolecules such as enzymes with nanoparticles has much to offer for creating next generation synergistically functional bionanomaterials. However, almost nothing is known about how these two disparate components interact at this critical biomolecular-materials interface to give rise to improved activity and emergent properties. Here we examine how the nanoparticle surface can influence and increase localized enzyme activity using a designer experimental system consisting of trypsin proteolysis acting on peptide-substrates displayed around semiconductor quantum dots (QDs). To minimize the complexity of analyzing this system, only the chemical nature of the QD surface functionalizing ligands were modified. This was accomplished by synthesizing a series of QD ligands that were either positively or negatively charged, zwitterionic, neutral, and with differing lengths. The QDs were then assembled with different ratios of dye-labeled peptide substrates and exposed to trypsin giving rise to progress curves that were monitored by Förster resonance energy transfer (FRET). The resulting trypsin activity profiles were analyzed in the context of detailed molecular dynamics simulations of key interactions occurring at this interface. Overall, we find that a combination of factors can give rise to a localized activity that was 35-fold higher than comparable freely diffusing enzyme-substrate interactions. Contributing factors include the peptide substrate being prominently displayed extending from the QD surface and not sterically hindered by the longer surface ligands in conjunction with the presence of electrostatic and other productive attractive forces between the enzyme and the QD surface. An intimate understanding of such critical interactions at this interface can produce a set of guidelines that will allow the rational design of next generation high-activity bionanocomposites and theranostics.