Colloidal quantum dots are desirable for a broad range of applications from biosensing to in vivo imaging due to their high luminosity and large surface areas available for bioconjugation. QDs are highly effective as fluorescent donors for Förster resonance energy transfer (FRET) and can be conjugated with multiple acceptors to enhance FRET efficiency and increase on/off ratios for sensing applications. We have demonstrated an efficient method for conjugating nucleic acids to QDs using chimeric peptide-peptide nucleic acids (peptide-PNA), and here we have characterized QD-FRET reporters assembled with this technique for use in CRISPR/Cas nuclease assays to identify factors which could limit the sensitivity of such reporters in practice.
DNA-directed assembly of gold nanoparticles into precise two- and three-dimensional patterns has enabled bold advances in probing their optical properties such as the local enhancement in their surface plasmon resonance. DNA nanostructures synthesized using the principles of DNA origami have been programmed to contain unique capture sites for positioning metal nanoparticles in diverse geometries for applications in biosensing, therapy, and miniature electronics. However, to enable scalability beyond simple 2-3 nanoparticle architectures, it is important to understand the requirement for orthogonal capture sequences for attaching more than a single gold nanoparticle on a DNA nanostructure. In this work, we sought to assemble an angular gold nanorod-nanosphere-nanorod pattern on a DNA origami triangle with multiple capture sites utilizing a common capture sequence. Results indicate that gold nanospheres preferentially bound to all the capture sites on the DNA origami triangle and prevented attachment of gold nanorods. This suggests that requirement for orthogonal capture sites is correlated with the physical properties of the individual nanoparticle such as shape and size.
The inherent ability of gold nanoparticles (AuNPs) to transduce light energy into heat, coupled with their ease of bioconjugation has made them a powerful tool potentially capable of controlling biological activity. When combined with ultra-short pulses of light and the proper experimental conditions, AuNPs are capable of heating their local environment without increasing the bulk solution temperature. Gene therapy and siRNA delivery have emerged as promising applications for localized heating of AuNPs and as such, a number of different groups have used light to trigger the release of nucleic acids from the surface of AuNPs. While successful nucleic acid release is universally demonstrated in the literature, the mechanism of release varies between reports. Specifically, the reported release mechanism is either: 1) the thermal denaturing of a nucleic acid duplex and release of a “single stranded” nucleic acid into solution; 2) the cleavage of the prototypical gold-thiol bond used to tether the nucleic acid duplex to the surface, resulting in the release of the complete nucleic acid duplex; or 3) a combination of both. Due to the complex parameter space in these experimental systems (AuNP size/shape/composition, laser energy density/repetition rate/pulse width) it is not surprising that the reported release mechanisms differ. Here, we utilize examples from the literature in order to identify the key parameters that dictate the release mechanism of nucleic acids on AuNPs in an attempt to further a comprehensive understanding of this process.
Time-gated Förster resonance energy transfer (TR-FRET) introduces a time-gate before the detection of the fluorescence spectra or photon count. If the donor is sufficiently long-lived TR-FRET allows for any initial acceptor sensitization to decay before the measurement. TR-FRET in the μs range is particularly advantageous for small molecule assays as it eliminates background fluorescence from screening compounds, which typically have ns lifetimes. The sensor we developed utilizes Terbium (Tb)-labeled antibodies (Ab) that selectively recognizes adenosine diphosphate (ADP). The Tb emitters have fluorescence lifetimes on the ms scale, making them excellent candidates for TR-FRET donors. In an attempt to increase the FRET signal we utilized a semiconductor quantum dot (QD) as an acceptor. The QD presented an ADP modified His6-peptide conjugated to its surface via self-assembly metal-affinity coordination, which bound the Tb labeled Ab to the QD surface. QDs have large extinction coefficients, broad absorption, brightness, and sharp emission peaks, optimal for sensitive and multiplexed detection. By using a QD acceptor the Förster radius was increased by approximately 2 nm as compared to traditional organic dyes. We were able to demonstrate a Tb-to-QD based TR-FRET bioassay for broadly applicable ADP sensing, working at nM concentrations for sensor, analyte, and enzyme. Quantitative values were obtained for the kinetics of a model enzyme (glucokinase). The specific sensor was also capable of discriminating enzyme inhibitor capabilities of structurally similar compounds. The strategy of using modified peptides to present antibody epitopes on QD surfaces is readily transferable to other assays.
The development of dynamic DNA nanostructures has opened the door to a wide variety of applications including sensing and information processing. DNA based molecular logic devices (MLDs) are DNA structures that have the ability to sense multiple inputs or “targets”, autonomously process the absence or presence of targets, and provide an output signal indicating the logic state of the system. As DNA is readily functionalized with fluorescent molecules, fluorophores can be strategically placed on MLDs so that the Förster resonance energy transfer efficiencies between the fluorophores are modulated when the DNA structure undergoes rearrangement. Consequently, the fluorescent signal of the dyes can be used as an output that provides the current logic state of the system. Although still in their elementary phase, MLDs have proved to be a promising modality for sensing multiple nanoscale targets, especially nucleic acids. Here, we review the development of multifluorophore MLDs and utilize examples from the literature and our own work to highlight their potential capabilities.
KEYWORDS: Light harvesting, Fluorescence resonance energy transfer, Nanostructures, DNA nanotechnology, DNA photonics, Bionanoengineering, Biophotonic applications
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.
KEYWORDS: Fluorescence resonance energy transfer, Light harvesting, Nanostructures, Energy transfer, Resonance energy transfer, Molecular photonics, Luminescence, Energy harvesting, Dendrimers, Molecules
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.
KEYWORDS: Sensors, Quantum dots, Fluorescence resonance energy transfer, Nanosensors, Luminescence, Resonance energy transfer, Nanoparticles, Systems modeling, Calibration, Signal detection
Nanosensors employing quantum dots (QDs) and enzyme substrates with fluorescent moieties offer tremendous promise for disease surveillance/diagnostics and as high-throughput co-factor assays. Advantages of QDs over other nanoscaffolds include their small size and inherent photochemical properties such as size tunable fluorescence, ease in attaching functional moieties, and resistance to photobleaching. These properties make QDs excellent Förster Resonance Energy Transfer (FRET) donors; well-suited for rapid, optical measurement applications. We report enzyme sensors designed with a single FRET donor, the QD donor acting as a scaffold to multiple substrates or acceptors. The QD-sensor follows the concrete activity of the enzyme, as compared to the most common methodologies that quantify the enzyme amount or its mRNA precursor. As the sensor reports on the enzyme activity in real-time we can actively follow the kinetics of the enzyme. Though classic Michaelis-Menten (MM) parameters can be obtained to describe the activity. In the course of these experiments deviations, both decreasing and increasing the kinetics, from the common MM model were observed upon close examinations. From these observations additional experiments were undertaken to understand the varying mechanisms. Different enzymes can present different deviations depending on the chosen target, e.g. trypsin appears to present a positive hopping mechanism while collagenase demonstrates a QD caused reversible inhibition.
KEYWORDS: Molecular photonics, Energy transfer, Fluorescence resonance energy transfer, Energy efficiency, Luminescence, Energy harvesting, Photosynthesis, Resonance energy transfer, Nanostructures, Anisotropy
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.
Enzymes are important players in multiple applications, be it bioremediation, biosynthesis, or as reporters. The business of catalysis and inhibition of enzymes is a multibillion dollar industry and understanding the kinetics of commercial enzymes can have a large impact on how these systems are optimized. Recent advances in nanotechnology have opened up the field of nanoparticle (NP) and enzyme conjugates and two principal architectures for NP conjugate systems have been developed. In the first example the enzyme is bound to the NP in a persistent manner, here we find that key factors such as directed enzyme conjugation allow for enhanced kinetics. Through controlled comparative experiments we begin to tease out specific mechanisms that may account for the enhancement. The second system is based on dynamic interactions of the enzymes with the NP. The enzyme substrate is bound to the NP and the enzyme is free in solution. Here again we find that there are many variables , such as substrate positioning and NP selection, that modify the kinetics.
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