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Cite this: DOI: 10.1039/c5nr00828j
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An enzymatically-sensitized sequential and concentric energy transfer relay self-assembled around semiconductor quantum dots† Anirban Samanta,a,b Scott A. Walper,a Kimihiro Susumu,c,d Chris L. Dwyere and Igor L. Medintz*a The ability to control light energy within de novo nanoscale structures and devices will greatly benefit their continuing development and ultimate application. Ideally, this control should extend from generating the light itself to its spatial propagation within the device along with providing defined emission wavelength(s), all in a stand-alone modality. Here we design and characterize macromolecular nanoassemblies consisting of semiconductor quantum dots (QDs), several differentially dye-labeled peptides and the enzyme luciferase which cumulatively demonstrate many of these capabilities by engaging in multiplesequential energy transfer steps. To create these structures, recombinantly-expressed luciferase and the dye-labeled peptides were appended with a terminal polyhistidine sequence allowing for controlled ratiometric self-assembly around the QDs via metal-affinity coordination. The QDs serve to provide multiple roles in these structures including as central assembly platforms or nanoscaffolds along with acting as a potent energy harvesting and transfer relay. The devices are activated by addition of coelenterazine H substrate which is oxidized by luciferase producing light energy which sensitizes the central 625 nm emitting QD acceptor by bioluminescence resonance energy transfer (BRET). The sensitized QD, in turn, acts as a relay and transfers the energy to a first peptide-labeled Alexa Fluor 647 acceptor dye displayed on its surface. This dye then transfers energy to a second red-shifted peptide-labeled dye acceptor on the QD surface through a second concentric Förster resonance energy transfer (FRET) process. Alexa Fluor 700 and Cy5.5 are both tested in the role of this terminal FRET acceptor. Photophysical analysis of spectral profiles
Received 4th February 2015, Accepted 4th March 2015
from the resulting sequential BRET–FRET–FRET processes allow us to estimate the efficiency of each of
DOI: 10.1039/c5nr00828j
the transfer steps. Importantly, the efficiency of each step within this energy transfer cascade can be controlled to some extent by the number of enzymes/peptides displayed on the QD. Further optimization of
www.rsc.org/nanoscale
the energy transfer process(es) along with potential applications of such devices are finally discussed.
1.
Introduction
The last decades have seen the tremendous development of an impressive array of nanomaterials each of which display unique and many times previously unavailable optical or physico-
a Center for Bio/Molecular Science and Engineering, Code 6900, U. S. Naval Research Laboratory, Washington, DC 20375 USA. E-mail: [email protected] b College of Science, George Mason University, Fairfax, VA 22030 USA c Optical Sciences Division, Code 5600, U.S. Naval Research Laboratory, Washington, DC 20375 USA d Sotera Defense Solutions, Columbia, MD 21046 USA e Department of Electrical and Computer Engineering, Duke University, Durham, NC 27708 USA † Electronic supplementary information (ESI) available: This material includes control experimental data and select deconvoluted spectra. See DOI: 10.1039/ c5nr00828j
This journal is © The Royal Society of Chemistry 2015
chemical properties.1–5 Equally impressive is the diversity of these materials which range from inorganic nanoparticles such as colloidal semiconductor quantum dots (QDs) and noble metal clusters to carbon allotropes such as graphene or nanodiamonds and even biologically-derived materials such as modified viral particles and de novo DNA structures.1–5 As the synthesis, properties, and especially understanding of these new materials continuously improves, one of the next great challenges is to begin integrating these new “building blocks” along with other synthetic or naturally occurring (nano)materials into emergent systems to create new functional systems by building with these parts from the bottom up. One potent functionality with the ability to enable many different types of nanosystems across a broad application space will be the inherent ability to controllably access, convert and propagate light energy in new and unique ways. In terms of available nanomaterials, luminescent semiconductor nanocrystals or
Nanoscale
View Article Online
Published on 25 March 2015. Downloaded by Northwestern University on 25/03/2015 19:46:50.
Paper
QDs have perhaps the most versatility to offer within both abiotic and biological nanoscale photonic heterostructures aimed at utilizing light energy. QDs inherently offer many unique spectroscopic capabilities including foremost the ability to tune their narrow-symmetric photoluminescence (PL) as a function of size and/or constituent semiconducting materials. QDs are also characterized by broad absorption spectra, large effective Stokes shifts, high quantum yields and strong resistance to photo- and chemical degradation.6–10 Cumulatively, this has led to their growing use as a fluorescent probe for numerous biological applications such as immunoassays along with cellular and in vivo imaging.6–11 These properties also allow QDs to function as versatile Förster resonance energy transfer (FRET) donors with a cumulatively unique set of capabilities including: the ability to be excited at an acceptor’s absorption minima thus limiting direct acceptor excitation; their emission can be tuned to optimize spectral overlap with a given acceptor; to engage in multiplex FRET; and, by virtue of their large two-photon (2P) action cross sections,10 to be specifically engaged as a donor within a 2P FRET configuration.6,12 Additionally, the ability to controllably array multiple (biologically-attached) FRET acceptors around the central QD donor allows for a proportional increase in FRET acceptor cross section which, in turn, allows for control over or “tuning” of relative FRET efficiency within the construct based on the ratio or valence of acceptor displayed on the QD surface.6 QD utility as a FRET acceptor is slightly more complicated by these same properties and usually requires a long-lifetime donor (e.g., lanthanide chelate) to overcome direct QD excitation coupled with its long excited state lifetime (10–50 ns versus 20 copies of this ∼37 kDa protein should easily assemble, i.e., without steric packing problems, to these QDs given their hard diameter of 9.2 ± 0.8 nm.32,55 Similarly, these QDs should be able to accommodate >100 copies of the peptides used. The ability of Luc9 to sensitize the 625 QDs was next examined. Increasing ratios of enzyme ranging from 0.5 up to 12 were preassembled on the QDs (final QD concentration 25 nM, final Luc9 concentration of 12.5–300 nM for ratios of 0.5 up to 12 per QD), placed in a cuvette, Coel substrate added (typically at least 500-fold excess over enzyme concentration) and emission spectra collected at 1 min and 4.5 min after substrate addition, see Fig. 2. Since Luc9 hydrolysis requires O2, which has low solubility in water (
Published on 25 March 2015. Downloaded by Northwestern University on 25/03/2015 19:46:50.
PAPER
Cite this: DOI: 10.1039/c5nr00828j
View Journal
An enzymatically-sensitized sequential and concentric energy transfer relay self-assembled around semiconductor quantum dots† Anirban Samanta,a,b Scott A. Walper,a Kimihiro Susumu,c,d Chris L. Dwyere and Igor L. Medintz*a The ability to control light energy within de novo nanoscale structures and devices will greatly benefit their continuing development and ultimate application. Ideally, this control should extend from generating the light itself to its spatial propagation within the device along with providing defined emission wavelength(s), all in a stand-alone modality. Here we design and characterize macromolecular nanoassemblies consisting of semiconductor quantum dots (QDs), several differentially dye-labeled peptides and the enzyme luciferase which cumulatively demonstrate many of these capabilities by engaging in multiplesequential energy transfer steps. To create these structures, recombinantly-expressed luciferase and the dye-labeled peptides were appended with a terminal polyhistidine sequence allowing for controlled ratiometric self-assembly around the QDs via metal-affinity coordination. The QDs serve to provide multiple roles in these structures including as central assembly platforms or nanoscaffolds along with acting as a potent energy harvesting and transfer relay. The devices are activated by addition of coelenterazine H substrate which is oxidized by luciferase producing light energy which sensitizes the central 625 nm emitting QD acceptor by bioluminescence resonance energy transfer (BRET). The sensitized QD, in turn, acts as a relay and transfers the energy to a first peptide-labeled Alexa Fluor 647 acceptor dye displayed on its surface. This dye then transfers energy to a second red-shifted peptide-labeled dye acceptor on the QD surface through a second concentric Förster resonance energy transfer (FRET) process. Alexa Fluor 700 and Cy5.5 are both tested in the role of this terminal FRET acceptor. Photophysical analysis of spectral profiles
Received 4th February 2015, Accepted 4th March 2015
from the resulting sequential BRET–FRET–FRET processes allow us to estimate the efficiency of each of
DOI: 10.1039/c5nr00828j
the transfer steps. Importantly, the efficiency of each step within this energy transfer cascade can be controlled to some extent by the number of enzymes/peptides displayed on the QD. Further optimization of
www.rsc.org/nanoscale
the energy transfer process(es) along with potential applications of such devices are finally discussed.
1.
Introduction
The last decades have seen the tremendous development of an impressive array of nanomaterials each of which display unique and many times previously unavailable optical or physico-
a Center for Bio/Molecular Science and Engineering, Code 6900, U. S. Naval Research Laboratory, Washington, DC 20375 USA. E-mail: [email protected] b College of Science, George Mason University, Fairfax, VA 22030 USA c Optical Sciences Division, Code 5600, U.S. Naval Research Laboratory, Washington, DC 20375 USA d Sotera Defense Solutions, Columbia, MD 21046 USA e Department of Electrical and Computer Engineering, Duke University, Durham, NC 27708 USA † Electronic supplementary information (ESI) available: This material includes control experimental data and select deconvoluted spectra. See DOI: 10.1039/ c5nr00828j
This journal is © The Royal Society of Chemistry 2015
chemical properties.1–5 Equally impressive is the diversity of these materials which range from inorganic nanoparticles such as colloidal semiconductor quantum dots (QDs) and noble metal clusters to carbon allotropes such as graphene or nanodiamonds and even biologically-derived materials such as modified viral particles and de novo DNA structures.1–5 As the synthesis, properties, and especially understanding of these new materials continuously improves, one of the next great challenges is to begin integrating these new “building blocks” along with other synthetic or naturally occurring (nano)materials into emergent systems to create new functional systems by building with these parts from the bottom up. One potent functionality with the ability to enable many different types of nanosystems across a broad application space will be the inherent ability to controllably access, convert and propagate light energy in new and unique ways. In terms of available nanomaterials, luminescent semiconductor nanocrystals or
Nanoscale
View Article Online
Published on 25 March 2015. Downloaded by Northwestern University on 25/03/2015 19:46:50.
Paper
QDs have perhaps the most versatility to offer within both abiotic and biological nanoscale photonic heterostructures aimed at utilizing light energy. QDs inherently offer many unique spectroscopic capabilities including foremost the ability to tune their narrow-symmetric photoluminescence (PL) as a function of size and/or constituent semiconducting materials. QDs are also characterized by broad absorption spectra, large effective Stokes shifts, high quantum yields and strong resistance to photo- and chemical degradation.6–10 Cumulatively, this has led to their growing use as a fluorescent probe for numerous biological applications such as immunoassays along with cellular and in vivo imaging.6–11 These properties also allow QDs to function as versatile Förster resonance energy transfer (FRET) donors with a cumulatively unique set of capabilities including: the ability to be excited at an acceptor’s absorption minima thus limiting direct acceptor excitation; their emission can be tuned to optimize spectral overlap with a given acceptor; to engage in multiplex FRET; and, by virtue of their large two-photon (2P) action cross sections,10 to be specifically engaged as a donor within a 2P FRET configuration.6,12 Additionally, the ability to controllably array multiple (biologically-attached) FRET acceptors around the central QD donor allows for a proportional increase in FRET acceptor cross section which, in turn, allows for control over or “tuning” of relative FRET efficiency within the construct based on the ratio or valence of acceptor displayed on the QD surface.6 QD utility as a FRET acceptor is slightly more complicated by these same properties and usually requires a long-lifetime donor (e.g., lanthanide chelate) to overcome direct QD excitation coupled with its long excited state lifetime (10–50 ns versus 20 copies of this ∼37 kDa protein should easily assemble, i.e., without steric packing problems, to these QDs given their hard diameter of 9.2 ± 0.8 nm.32,55 Similarly, these QDs should be able to accommodate >100 copies of the peptides used. The ability of Luc9 to sensitize the 625 QDs was next examined. Increasing ratios of enzyme ranging from 0.5 up to 12 were preassembled on the QDs (final QD concentration 25 nM, final Luc9 concentration of 12.5–300 nM for ratios of 0.5 up to 12 per QD), placed in a cuvette, Coel substrate added (typically at least 500-fold excess over enzyme concentration) and emission spectra collected at 1 min and 4.5 min after substrate addition, see Fig. 2. Since Luc9 hydrolysis requires O2, which has low solubility in water (
