ABSTRACT There is considerable research in the area of manipulating light below the diffraction limit, with potential applications ranging from information processing to light-harvesting. In such work, a common problem is a lack of... more
ABSTRACT There is considerable research in the area of manipulating light below the diffraction limit, with potential applications ranging from information processing to light-harvesting. In such work, a common problem is a lack of efficiency associated with non-radiative losses, e.g., ohmic loss in plasmonic structures. From this point of view, one attractive method for sub-wavelength light manipulation is to use Förster resonance energy transfer (FRET) between chromophores. Although most current work does not show high efficiency, biology suggests that this approach could achieve very high efficiency. In order to achieve this goal, the geometry and spacing of the chromophores must be optimized. For this, DNA provides an easy means for the self-assembly of these complex structures. With well established ligation chemistries, it is possible to create facile hierarchical assemblies of quantum dots (QDs) and organic dyes using DNA as the platform. These nanostructures range from simple linear wires to complex 3-dimensional structures all of which can be self-assembled around a central QD. The efficiency of the system can then be tuned by changing the spacing between chromophores, changing the DNA geometry such that the donor to acceptor ratio changes, or changing the number of DNA structures that are self-assembled around the central QD. By exploring these variables we have developed a flexible optical system for which the efficiency can be both controlled and optimized.
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DNA scaffolds provide a means to precisely organize chromophores into large biomimetic exciton networks and direct energy transport for nanoscale sensing and light‐harvesting applications. Here, a functional building block of minimal... more
DNA scaffolds provide a means to precisely organize chromophores into large biomimetic exciton networks and direct energy transport for nanoscale sensing and light‐harvesting applications. Here, a functional building block of minimal complexity that maximizes the Förster resonance energy transfer (FRET) efficiency is sought. Using a model system consisting of three FRET steps in a 4‐dye cascade: Cy3→Cy3.5→Cy5→Cy5.5, we evaluate how this building block employs multiple interacting versus redundant FRET pathways. Variants of a dual rail design, where one or two copies of each dye are aligned in rigid linear parallel rows, are compared to a split rail format, where varying degrees of spacing are introduced between the rows. The FRET processes are assessed via steady‐state, time‐resolved, and single‐molecule spectroscopy. Experiments and simulation reveal the dual rail design as more efficient than the split rail and suggest the design principle that efficient FRET networks must balance the increase in FRET rate from multiple interacting pathways with undesirable fluorescence quenching between dyes in close proximity. Hybrid fluorophore combinations are identified as a strategy to mitigate this quenching, leading to optimized dual rails capable of 50% end‐to‐end efficiency. These insights can help guide the design of functional photonic wires based on DNA scaffolds.
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DNA scaffolds offer advantages for biomolecular devices capable of generating and controlling energy flow. Through attachment of the bioluminescent protein luciferase and precise positioning of organic fluorophores, these structures form... more
DNA scaffolds offer advantages for biomolecular devices capable of generating and controlling energy flow. Through attachment of the bioluminescent protein luciferase and precise positioning of organic fluorophores, these structures form efficient self‐illuminating energy transfer cascades. Luciferase initiates a bioluminescent resonance energy transfer step, which is then propagated by Förster resonance energy transfer (FRET) through the rest of the structure. Two related DNA dendrimeric nanostructures are investigated; the first funnels energy inward toward the center, while the second radiates it outward. When attached to the DNA dendrimer, luciferase bioluminescence is harnessed by an initial AlexaFluor 488 dye and transferred via a series of FRET steps through Cy3, Cy3.5, and Cy5 dyes – to a terminal Cy5.5 acceptor. In the inward funneling construct, the luciferase is displayed on the dendrimer periphery and uses two donor fluorophores for each subsequent acceptor. The outward funneling construct anchors luciferase at the core and energies of successive donor fluorophores are captured by two acceptor dyes. The inward configuration yields end‐to‐end and anywhere‐to‐end efficiencies of 16% and 25%, respectively, while the outward configuration approaches 25% and 50%, respectively. The performance of each construct is detailed and potential applications of these self‐illuminating biophotonic assemblies are discussed.
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Research Interests: Biochemistry, Genetics, Biophysics, Materials Science, Molecular Biology, and 15 moreBiotechnology, Plant Biology, Fluorescence, Response, Nanotechnology, Infectious Diseases, Space Science, Medicine, Manipulation, Biosensor, Mechanism, Applications of Alcohol Oxidase In Biosensing, Dendrimer, Acceptor, and Transfer efficiency
Research Interests: Materials Science, Electrochemistry, Transmission Electron Microscopy, Nanotechnology, Atomic Force Microscopy, and 13 moreMedicine, Multidisciplinary, Fluorescence Resonance Energy Transfer, Dna Nanotechnology, DNA, Biosensor, Light, Nanostructures, Nanostructure, Base Sequence, Nucleic Acid Conformation, Molecular probes, and DNA origami
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DNA scaffolds provide a means to precisely organize chromophores into large biomimetic exciton networks and direct energy transport for nanoscale sensing and light‐harvesting applications. Here, a functional building block of minimal... more
DNA scaffolds provide a means to precisely organize chromophores into large biomimetic exciton networks and direct energy transport for nanoscale sensing and light‐harvesting applications. Here, a functional building block of minimal complexity that maximizes the Förster resonance energy transfer (FRET) efficiency is sought. Using a model system consisting of three FRET steps in a 4‐dye cascade: Cy3→Cy3.5→Cy5→Cy5.5, we evaluate how this building block employs multiple interacting versus redundant FRET pathways. Variants of a dual rail design, where one or two copies of each dye are aligned in rigid linear parallel rows, are compared to a split rail format, where varying degrees of spacing are introduced between the rows. The FRET processes are assessed via steady‐state, time‐resolved, and single‐molecule spectroscopy. Experiments and simulation reveal the dual rail design as more efficient than the split rail and suggest the design principle that efficient FRET networks must balance the increase in FRET rate from multiple interacting pathways with undesirable fluorescence quenching between dyes in close proximity. Hybrid fluorophore combinations are identified as a strategy to mitigate this quenching, leading to optimized dual rails capable of 50% end‐to‐end efficiency. These insights can help guide the design of functional photonic wires based on DNA scaffolds.
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Because of their ease of design and assembly, DNA scaffolds provide a valuable means for organizing fluorophores into complex light harvesting antennae. However, as the size and complexity of the DNA–fluorophore network grows, it can be... more
Because of their ease of design and assembly, DNA scaffolds provide a valuable means for organizing fluorophores into complex light harvesting antennae. However, as the size and complexity of the DNA–fluorophore network grows, it can be difficult to fully understand energy transfer properties because of the large number of dipolar interactions between fluorophores. Here, we investigate simple DNA–fluorophore networks that represent elements of the more complex networks and provide insight into the Forster Resonance Energy Transfer (FRET) processes in the presence of multiple pathways. These FRET networks consist of up to two Cy3 donor fluorophores and two Cy3.5 acceptor fluorophores that are linked to a rigid dual-rail DNA scaffold with short interfluorophore separation corresponding to 10 DNA base pairs (∼34 A). This configuration results in five FRET pathways: four hetero-FRET and one homo-FRET pathway. The FRET properties are characterized using a combination of steady-state and time-resolved spectrosc...
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Research Interests: Chemistry, Nanocomposites, Nanotechnology, Fluorescent Dyes and Reagents, Medicine, and 14 moreMultidisciplinary, Fluorescence Resonance Energy Transfer, DNA, Quantum Dots, Biosensor, Biotin, Streptavidin, Quantum Dot, Molecular Conformation, Base Sequence, Histidine, Polyethylene Glycols, fluorescent dyes, and DNA origami
The growing maturity of DNA-based architectures has raised considerable interest in applying them to create photoactive light harvesting and sensing devices. Toward optimizing efficiency in such structures, resonant energy transfer was... more
The growing maturity of DNA-based architectures has raised considerable interest in applying them to create photoactive light harvesting and sensing devices. Toward optimizing efficiency in such structures, resonant energy transfer was systematically examined in a series of dye-labeled DNA duplexes where donor-acceptor separation was incrementally changed from 0 to 16 base pairs. Cyanine dyes were localized on the DNA using double phosphoramidite attachment chemistry. Steady state spectroscopy, single-pair fluorescence, time-resolved fluorescence, and ultrafast two-color pump-probe methods were utilized to examine the energy transfer processes. Energy transfer rates were found to be more sensitive to the distance between the Cy3 donor and Cy5 acceptor dye molecules than efficiency measurements. Picosecond energy transfer and near-unity efficiencies were observed for the closest separations. Comparison between our measurements and the predictions of Förster theory based on structural modeling of the dye-labeled DNA duplex suggest that the double phosphoramidite linkage leads to a distribution of intercalated and nonintercalated dye orientations. Deviations from the predictions of Förster theory point to a failure of the point dipole approximation for separations of less than 10 base pairs. Interactions between the dyes that alter their optical properties and violate the weak-coupling assumption of Förster theory were observed for separations of less than four base pairs, suggesting the removal of nucleobases causes DNA deformation and leads to enhanced dye-dye interaction.
Research Interests: Engineering, Biochemistry, Microbiology, Biophysics, Chemistry, and 15 morePhotochemistry, Fluorescence, Prediction, Fluorescent Dyes and Reagents, Medicine, Fluorescence Resonance Energy Transfer, DNA, Fo, Physical sciences, CHEMICAL SCIENCES, Energy Transfer, Nucleic Acid Conformation, Acceptor, Cyanine Dyes, and fluorescent dyes
The progress within the field of DNA nanotechnology has shown DNA to be an ideal material for the self-assembly of complex two-dimensional and three-dimensional structures. In addition to forming a wide range of structural geometries, DNA... more
The progress within the field of DNA nanotechnology has shown DNA to be an ideal material for the self-assembly of complex two-dimensional and three-dimensional structures. In addition to forming a wide range of structural geometries, DNA has been demonstrated as an exemplary scaffold. In this work we utilize this scaffolding ability to create photonic DNA switches that respond to both DNA and enzymatic inputs and produce complex logic based outputs.
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DNA is a biocompatible scaffold that allows for the design of a variety of nanostructures, from straightforward double stranded DNA to more complex DNA origami and 3-D structures. By modifying the structures, with dyes, nanoparticles, or... more
DNA is a biocompatible scaffold that allows for the design of a variety of nanostructures, from straightforward double stranded DNA to more complex DNA origami and 3-D structures. By modifying the structures, with dyes, nanoparticles, or enzymes, they can be used to create light harvesting and energy transfer systems. We have focused on using Förster resonance energy transfer (FRET) between organic fluorophores separated with nanometer precision based on the DNAs defined positioning. Using FRET theory we can control the direction of the energy flow and optimize the design parameters to increase the systems efficiency. The design parameters include fluorophore selection, separation, number, and orientation among others. Additionally the use of bioluminescence resonance energy transfer (BRET) allowed the use of chemical energy, as opposed to photonic, to activate the systems. Here we discuss a variety of systems, such as the longest reported DNA-based molecular photonic wires (> 30 nm), dendrimeric light harvesting systems, and semiconductor nanocrystals integrated systems where they act as both scaffold and antennae for the original excitation. Using a variety of techniques, a comparison of different types of structures as well as heterogeneous vs. homogenous FRET was realized.
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Light harvesting quantum dot-dye-labeled DNA dendrimer structures are assembled yielding end-to-end energy transfer efficiencies approaching 25% over 4 FRET steps.
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Molecular photonic wires (MPWs) precisely position dyes using structural DNA methodologies where they exploit Förster resonance energy transfer (FRET) to direct photonic energy over nm distances with potential applications in light... more
Molecular photonic wires (MPWs) precisely position dyes using structural DNA methodologies where they exploit Förster resonance energy transfer (FRET) to direct photonic energy over nm distances with potential applications in light harvesting, biosensing, and molecular electronics. Although versatile, the number of donor–acceptor dye pairs available and the downhill nature of FRET combine to limit the size and efficiency of current MPWs. HomoFRET between identical dyes should provide zero energy loss but at the cost of random transfer directionality. Here, it has been investigated what HomoFRET has to offer as a means to extend MPWs. Steady‐state‐, lifetime‐, and fluorescence anisotropy measurements along with mathematical models are utilized to experimentally examine various 3‐, 4‐, and 5‐dye MPW constructs containing from 1 to 6 HomoFRET repeat sections. Results show that HomoFRET can be extended up to 6 repeat dyes/5 steps with only a ≈55% energy transfer efficiency decrease while doubling the longest MPW length to a remarkable 30 nm. Critically, analogous constructs lacking the HomoFRET portion are unable to deliver any energy over the same lengths. Even with nondirectionality, the introduction of a repeated‐optimized HomoFRET transfer dye is preferable compared to additional less efficient dye species. HomoFRET further provides the benefit of having a higher energy output.
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ABSTRACT The “spectroscopic ruler” based on fluorescence resonance energy transfer (FRET) is explored as a method for detailed structural characterization of DNA nanostructures in solution. The approach is most directly useful for... more
ABSTRACT The “spectroscopic ruler” based on fluorescence resonance energy transfer (FRET) is explored as a method for detailed structural characterization of DNA nanostructures in solution. The approach is most directly useful for assessing the positional relationships among chromophores organized by the DNA, but it can also be used to characterize the geometry and kinematics of the DNA scaffold itself. By accumulating data for the distances separating various donor-acceptor pairs, and correlating them with the expected distances, one can quantify the shape and deformability of the structure. A 8x16nm “mini-origami” rectangle is used as the model test structure and the dye-pairs are chosen to investigate anisotropy in the origami’s mechanical properties. Not unexpectedly, our analysis finds a strong anisotropy in the stiffness, with the measured spacing across the origami weave deviating much more from expectation than the spacing aligned along the weave pattern.
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DNA as a structural nanomaterial demonstrates great potential as both an in vivo and in vitro designer platform for diagnostic and therapeutic medical use. Much of this work hinges on the ability of DNA to assemble into discrete,... more
DNA as a structural nanomaterial demonstrates great potential as both an in vivo and in vitro designer platform for diagnostic and therapeutic medical use. Much of this work hinges on the ability of DNA to assemble into discrete, controlled structures that interact with, or bind to, other inorganic materials such as nanoparticles or biological molecules which include, for example, drugs and proteins. For these functionalized structures to be most effective, the spatial accuracy of their assembly must be precisely monitored and controlled. Clearly, to design and implement all forms of these functionalized DNA structures, a full characterization will ultimately be a critical necessity. With the current array of characterization techniques available, it can be difficult to choose one specific method especially considering that the efficacy can depend on the type of structure and the final application and environment in which the structure will be used. A review of current methods used for the characterization of complex DNA nanostructures can provide us with a greater understanding of which structures and applications will benefit from specific techniques. More importantly, it can also yield an understanding of which characterization methods can be used in concert to provide a more in depth and integrated understanding of a particular construct as a whole. Comparative characterization may also provide information on the many subtleties and nuances that are to be expected in these complex systems. In this critical overview of available characterization methods, we examine the techniques currently in use for these purposes.
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The development of light harvesting systems for directed, efficient control of energy transfer at the biomolecular level has generated considerable interest in the past decade. Molecular fluorophores provide a straightforward mechanism... more
The development of light harvesting systems for directed, efficient control of energy transfer at the biomolecular level has generated considerable interest in the past decade. Molecular fluorophores provide a straightforward mechanism for determining nanoscale distance changes through Förster resonance energy transfer (FRET), and many systems seek to build off of this simple yet powerful principle to provide additional functionality. The use of DNA-based integrated biomolecular devices offer many unique advantages towards this end. DNA itself is an excellent engineering material – it is innately biocompatible, quickly and cheaply synthesized, and complex structures can be readily designed in silico. It also provides an excellent scaffold for the precise patterning of various biomolecules. Here, we discuss the systems that have been recently developed which add to this toolbox, including nanostructural dye patterning, photonic wires, and the incorporation of alternative energy propagation modalities, such as semiconductor quantum dots (QD) and the bioluminescent protein luciferase. In particular, we explore the incorporation of luciferase into various nanostructural conformations, providing the capability to efficiently control energy flow directionality. We discuss the nature of this system, including unexpected spectral complexities, in the context of the field.
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We demonstrate that unstained DNA nanostructures can be chemically mapped by using aberration-corrected scanning transmission electron microscopy (STEM) with energy-dispersive spectroscopy (EDS). Key to this measurement is the use of... more
We demonstrate that unstained DNA nanostructures can be chemically mapped by using aberration-corrected scanning transmission electron microscopy (STEM) with energy-dispersive spectroscopy (EDS). Key to this measurement is the use of graphene supports whose reduced background scattering compared to thicker carbon supports allows EDS to image phosphorus and other elements in the DNA (or in attachments to the DNA) as well as to see the divalent cations used to stabilize the nanostructure. In addition, unlike in other EDS analyses, the chemical maps we obtain can be made quantitative in an absolute sense because each individual DNA nanostructure is in effect a very large macromolecule (∼4.7 MDa) with a known chemical composition. In this way, not only can STEM/EDS serve DNA nanotechnology as a characterization tool but also the DNA structures can function as molecular standards for improving mesoscale EDS techniques.
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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... more
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.
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Molecular photonic wires (MPWs) precisely position dyes using structural DNA methodologies where they exploit Förster resonance energy transfer (FRET) to direct photonic energy over nm distances with potential applications in light... more
Molecular photonic wires (MPWs) precisely position dyes using structural DNA methodologies where they exploit Förster resonance energy transfer (FRET) to direct photonic energy over nm distances with potential applications in light harvesting, biosensing, and molecular electronics. Although versatile, the number of donor–acceptor dye pairs available and the downhill nature of FRET combine to limit the size and efficiency of current MPWs. HomoFRET between identical dyes should provide zero energy loss but at the cost of random transfer directionality. Here, it has been investigated what HomoFRET has to offer as a means to extend MPWs. Steady‐state‐, lifetime‐, and fluorescence anisotropy measurements along with mathematical models are utilized to experimentally examine various 3‐, 4‐, and 5‐dye MPW constructs containing from 1 to 6 HomoFRET repeat sections. Results show that HomoFRET can be extended up to 6 repeat dyes/5 steps with only a ≈55% energy transfer efficiency decrease while doubling the longest MPW length to a remarkable 30 nm. Critically, analogous constructs lacking the HomoFRET portion are unable to deliver any energy over the same lengths. Even with nondirectionality, the introduction of a repeated‐optimized HomoFRET transfer dye is preferable compared to additional less efficient dye species. HomoFRET further provides the benefit of having a higher energy output.
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Molecular photonic wires (MPWs) present interesting applications in energy harvesting, artificial photosynthesis, and nano-circuitry. MPWs allow the directed movement of energy at the nanoscopic level. Extending the length of the energy... more
Molecular photonic wires (MPWs) present interesting applications in energy harvesting, artificial photosynthesis, and nano-circuitry. MPWs allow the directed movement of energy at the nanoscopic level. Extending the length of the energy transfer with a minimal loss in efficiency would overcome an important hurdle in allowing MPWs to reach their potential. We investigated Homogenous Förster Resonance Energy Transfer (HomoFRET) as a means to achieve this goal. We designed a simple, self-assembled DNA nanostructure with specifically placed dyes (Alexa488-Cy3-Cy3.5-Alexa647-Cy5.5) at a distance of 3.4 nm, a separation at which energy transfer should theoretically be very high. The input of the wire was at 466 nm with an output up to 697 nm. Different structures were studied where the Cy3.5 section of the MPW was extended from one to six repeats. We found that though the efficiency cost is not null, HomoFRET can be extended up to six repeat dyes with only a 22% efficiency loss when compared to a single step system. The advantage is that these six repeats created a MPW which was 17 nm longer, almost 2.5 times the initial length. To confirm the existence of HomoFRET between the Cy3.5 repeats fluorescence lifetime and fluorescence lifetime anisotropy was measured. Under these conditions we are able to demonstrate the energy transfer over a distance of 30.4 nm, with an end-to-end efficiency of 2.0%, by utilizing a system with only five unique dyes.
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ABSTRACT
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This paper reports design improvements to minimize parasitic reactions in bioMEMS for studying sequential, site-specific enzymatic reactions. Interconnect reservoirs were eliminated by employing aligners on prototype mold to guide... more
This paper reports design improvements to minimize parasitic reactions in bioMEMS for studying sequential, site-specific enzymatic reactions. Interconnect reservoirs were eliminated by employing aligners on prototype mold to guide packaging. Flow directions for in situ enzyme assembly and subsequent enzymatic reaction were separated by employing a cross-channel design. These improvements efficiently suppressed parasitic reactions by trapped enzyme in the interconnect reservoirs and non-specifically bound enzyme on microchannel walls.
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The progress within the field of DNA nanotechnology has shown DNA to be an ideal material for the self-assembly of complex two-dimensional and three-dimensional structures. In addition to forming a wide range of structural geometries, DNA... more
The progress within the field of DNA nanotechnology has shown DNA to be an ideal material for the self-assembly of complex two-dimensional and three-dimensional structures. In addition to forming a wide range of structural geometries, DNA has been demonstrated as an exemplary scaffold. In this work we utilize this scaffolding ability to create photonic DNA switches that respond to both DNA and enzymatic inputs and produce complex logic based outputs.
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Detecting, identifying and quantifying small molecules in bioMEMS are important to metabolic engineering applications, e.g. enzyme reaction pathways. We present a novel method for creating spatially localized sites for surface enhanced... more
Detecting, identifying and quantifying small molecules in bioMEMS are important to metabolic engineering applications, e.g. enzyme reaction pathways. We present a novel method for creating spatially localized sites for surface enhanced Raman spectroscopy (SERS) in completed microfluidic systems to enable in situ sensing of reaction products. When adenine, a prototype small molecule, is introduced into the microfluidic channel, its Raman spectrum is detected through SERS interaction at such sites. Furthermore, the chitosan surface is still available for chemical functionalization, thus creating the potential for in-situ measurement of enzymatic reaction products at the enzyme site itself.
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The development of light harvesting systems for directed, efficient control of energy transfer at the biomolecular level has generated considerable interest in the past decade. Molecular fluorophores provide a straightforward mechanism... more
The development of light harvesting systems for directed, efficient control of energy transfer at the biomolecular level has generated considerable interest in the past decade. Molecular fluorophores provide a straightforward mechanism for determining nanoscale distance changes through Förster resonance energy transfer (FRET), and many systems seek to build off of this simple yet powerful principle to provide additional functionality. The use of DNA-based integrated biomolecular devices offer many unique advantages towards this end. DNA itself is an excellent engineering material – it is innately biocompatible, quickly and cheaply synthesized, and complex structures can be readily designed in silico. It also provides an excellent scaffold for the precise patterning of various biomolecules. Here, we discuss the systems that have been recently developed which add to this toolbox, including nanostructural dye patterning, photonic wires, and the incorporation of alternative energy propa...
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DNA-based assemblies provide a simple and economical preparation method for molecular photonic wires (structures that capture and direct light with high efficiencies), through precise positioning of the molecular transfer components.... more
DNA-based assemblies provide a simple and economical preparation method for molecular photonic wires (structures that capture and direct light with high efficiencies), through precise positioning of the molecular transfer components. Multiple variables were studied to optimize these FRET based molecular photonic wires.