Multiplexed DNA and Protease Detection with Orthogonal Energy Transfer on a Single Quantum Dot Scaffolded Biosensor.

Almost all pathogens, whether viral or bacterial, utilize key proteolytic steps in their pathogenesis. The ability to detect a pathogen's genomic material along with its proteolytic activity represents one approach to identifying the pathogen and providing initial evidence of its viability. Here, we report on a prototype biosensor design assembled around a single semiconductor quantum dot (QD) scaffold that is capable of detecting both nucleic acid sequences and proteolytic activity by using orthogonal energy transfer (ET) processes. The sensor consists of a central QD assembled via peptidyl-PNA linkers with multiple DNA sequences that encode complements to genomic sequences originating from the Ebola, Influenza, and COVID-19 viruses, which we use as surrogate targets. These are hybridized to complement strands labeled with a terbium (Tb) chelate, AlexaFluor647 (AF647), and Cy5.5 dyes, giving rise to two potential FRET cascades: the first includes Tb → QD → AF647 → Cy5.5 (→ = ET step), which is detected in a time-gated modality, and QD → AF647 → Cy5.5, which is detected from direct excitation. The labeled DNA-displaying QD construct is then further assembled with a RuII-modified peptide, which quenches QD photoluminescence by charge transfer and is recognized by a protease to yield the full biosensor. Each of the labeled DNAs and peptides can be ratiometrically assembled to the QD in a controllable manner to tune each of the ET pathways. Addition of a given target DNA displaces its labeled complement on the QD, disrupting that FRET channel, while protease addition disrupts charge transfer quenching of the central QD scaffold and boosts its photoluminescence and FRET relay capabilities. Along with characterizing the ET pathways and verifying biosensing in both individual and multiplexed formats, we also demonstrate the ability of this construct to function in molecular logic and perform Boolean operations; this highlights the construct's ability to discriminate and transduce signals between different inputs or pathogens. The potential application space for such a sensor device is discussed.

Towards control of excitonic coupling in DNA-templated Cy5 aggregates: the principal role of chemical substituent hydrophobicity and steric interactions.

Understanding and controlling exciton coupling in dye aggregates has become a greater focus as potential applications such as coherent exciton devices, nanophotonics, and biosensing have been proposed. DNA nanostructure templates allow for a powerful modular approach. Using DNA Holliday junction (HJ) templates variations of dye combinations and precision dye positions can be rapidly assayed, as well as creating aggregates of dyes that could not be prepared (either due to excess or lack of solubility) through alternative means. Indodicarbocyanines (Cy5) have been studied in coupled systems due to their large transition dipole moment, which contributes to strong coupling. Cy5-R dyes were recently prepared by chemically modifying the 5,5'-substituents of indole rings, resulting in varying dye hydrophobicity/hydrophilicity, steric considerations, and electron-donating/withdrawing character. We utilized Cy5-R dyes to examine the formation and properties of 30 unique DNA templated homodimers. We find that in our system the sterics of Cy5-R dyes play the determining factor in orientation and coupling strength of dimers, with coupling strengths ranging from 50-138 meV. The hydrophobic properties of the Cy5-R modify the percentage of dimers formed, and have a secondary role in determining the packing characteristics of the dimers when sterics are equivalent. Similar to other reports, we find that positioning of the Cy5-R within the HJ template can favor particular dimer interactions, specifically oblique or H-type dimers.

Structural and optical variation of pseudoisocyanine aggregates nucleated on DNA substrates.

Coherently coupled pseudoisocyanine (PIC) dye aggregates have demonstrated the ability to delocalize electronic excitations and ultimately migrate excitons with much higher efficiency than similar designs where excitations are isolated to individual chromophores. Here, we report initial evidence of a new type of PIC aggregate, formed through heterogeneous nucleation on DNA oligonucleotides, displaying photophysical properties that differ significantly from previously reported aggregates. This new aggregate, which we call the super aggregate (SA) due to the need for elevated dye excess to form it, is clearly differentiated from previously reported aggregates by spectroscopic and biophysical characterization. In emission spectra, the SA exhibits peak narrowing and, in some cases, significant quantum yield variation, indicative of stronger coupling in cyanine dyes. The SA was further characterized with circular dichroism and atomic force microscopy observing unique features depending on the DNA substrate. Then by integrating an AlexaFluorTM647 (AF) dye as an energy transfer acceptor into the system, we observed mixed energy transfer characteristics using the different DNA. For example, SA formed with a rigid DNA double crossover tile (DX-tile) substrate resulted in AF emission sensitization. While SA formed with more flexible non-DX-tile DNA (i.e. duplex and single strand DNA) resulted in AF emission quenching. These combined characterizations strongly imply that DNA-based PIC aggregate properties can be controlled through simple modifications to the DNA substrate's sequence and geometry. Ultimately, we aim to inform rational design principles for future device prototyping. For example, one key conclusion of the study is that the high absorbance cross-section and efficient energy transfer observed with rigid substrates made for better photonic antennae, compared to flexible DNA substrates.

Tunable Electronic Structure via DNA-Templated Heteroaggregates of Two Distinct Cyanine Dyes.

Molecular excitons are useful for applications in light harvesting, organic optoelectronics, and nanoscale computing. Electronic energy transfer (EET) is a process central to the function of devices based on molecular excitons. Achieving EET with a high quantum efficiency is a common obstacle to excitonic devices, often owing to the lack of donor and acceptor molecules that exhibit favorable spectral overlap. EET quantum efficiencies may be substantially improved through the use of heteroaggregates-aggregates of chemically distinct dyes-rather than individual dyes as energy relay units. However, controlling the assembly of heteroaggregates remains a significant challenge. Here, we use DNA Holliday junctions to assemble homo- and heterotetramer aggregates of the prototypical cyanine dyes Cy5 and Cy5.5. In addition to permitting control over the number of dyes within an aggregate, DNA-templated assembly confers control over aggregate composition, i.e., the ratio of constituent Cy5 and Cy5.5 dyes. By varying the ratio of Cy5 and Cy5.5, we show that the most intense absorption feature of the resulting tetramer can be shifted in energy over a range of almost 200 meV (1600 cm-1). All tetramers pack in the form of H-aggregates and exhibit quenched emission and drastically reduced excited-state lifetimes compared to the monomeric dyes. We apply a purely electronic exciton theory model to describe the observed progression of the absorption spectra. This model agrees with both the measured data and a more sophisticated vibronic model of the absorption and circular dichroism spectra, indicating that Cy5 and Cy5.5 heteroaggregates are largely described by molecular exciton theory. Finally, we extend the purely electronic exciton model to describe an idealized J-aggregate based on Förster resonance energy transfer (FRET) and discuss the potential advantages of such a device over traditional FRET relays.

Tuning between Quenching and Energy Transfer in DNA-Templated Heterodimer Aggregates.

Molecular excitons, which propagate spatially via electronic energy transfer, are central to numerous applications including light harvesting, organic optoelectronics, and nanoscale computing; they may also benefit applications such as photothermal therapy and photoacoustic imaging through the local generation of heat via rapid excited-state quenching. Here we show how to tune between energy transfer and quenching for heterodimers of the same pair of cyanine dyes by altering their spatial configuration on a DNA template. We assemble "transverse" and "adjacent" heterodimers of Cy5 and Cy5.5 using DNA Holliday junctions. We find that the transverse heterodimers exhibit optical properties consistent with excitonically interacting dyes and fluorescence quenching, while the adjacent heterodimers exhibit optical properties consistent with nonexcitonically interacting dyes and disproportionately large Cy5.5 emission, suggestive of energy transfer between dyes. We use transient absorption spectroscopy to show that quenching in the transverse heterodimer occurs via rapid nonradiative decay to the ground state (∼31 ps) and that in the adjacent heterodimer rapid energy transfer from Cy5 to Cy5.5 (∼420 fs) is followed by Cy5.5 excited-state relaxation (∼700 ps). Accessing such drastically different photophysics, which may be tuned on demand for different target applications, highlights the utility of DNA as a template for dye aggregation.

Rapid DNA origami nanostructure detection and classification using the YOLOv5 deep convolutional neural network.

The intra-image identification of DNA structures is essential to rapid prototyping and quality control of self-assembled DNA origami scaffold systems. We postulate that the YOLO modern object detection platform commonly used for facial recognition can be applied to rapidly scour atomic force microscope (AFM) images for identifying correctly formed DNA nanostructures with high fidelity. To make this approach widely available, we use open-source software and provide a straightforward procedure for designing a tailored, intelligent identification platform which can easily be repurposed to fit arbitrary structural geometries beyond AFM images of DNA structures. Here, we describe methods to acquire and generate the necessary components to create this robust system. Beginning with DNA structure design, we detail AFM imaging, data point annotation, data augmentation, model training, and inference. To demonstrate the adaptability of this system, we assembled two distinct DNA origami architectures (triangles and breadboards) for detection in raw AFM images. Using the images acquired of each structure, we trained two separate single class object identification models unique to each architecture. By applying these models in sequence, we correctly identified 3470 structures from a total population of 3617 using images that sometimes included a third DNA origami structure as well as other impurities. Analysis was completed in under 20 s with results yielding an F1 score of 0.96 using our approach.

Understanding Self-Assembled Pseudoisocyanine Dye Aggregates in DNA Nanostructures and Their Exciton Relay Transfer Capabilities.

Progress has been made using B-form DNA duplex strands to template chromophores in ordered molecular aggregates known as J-aggregates. These aggregates can exhibit strong electronic coupling, extended coherent lifetimes, and long-range exciton delocalization under appropriate conditions. Certain cyanine dyes such as pseudoisocyanine (PIC) dye have shown a proclivity to form aggregates in specific DNA sequences. In particular, DX-tiles containing nonalternating poly(dA)-poly(dT) dinucleotide tracks (AT-tracks), which template noncovalent PIC dye aggregates, have been demonstrated to exhibit interesting emergent photonic properties. These DNA-based aggregates are referred to as J-bits for their similarity to J-aggregates. Here, we assemble multifluorophore DX-tile scaffolds which template J-bits into both contiguous and noncontiguous linear arrays. Our goal is to understand the relay capability of noncontiguous J-bit arrays and probe the effects that orientation and position have on the energy transfer between them. We find that linearly contiguous J-bits can relay excitons from an initial AlexaFluor 405 donor to a terminal AlexaFluor 647 acceptor across a distance of up to 16.3 nm. We observed a maximum increase in energy transfer of 41% in the shortest scaffold and an 11% increase in energy transfer across the maximum distance. However, in nonlinear arrays, exciton transfer is not detectable, even when off-axis J-bit-to-J-bit transfer distances were <2 nm. These results, in conjunction with the previous work on PIC-DNA systems, suggest that PIC-DNA-based systems may currently be limited to simple 1-D designs, which prevent isolating J-bits for enhanced energy-transfer characteristics until further understanding and improvements to the system can be made.

Femtosecond Laser Pulse Excitation of DNA-Labeled Gold Nanoparticles: Establishing a Quantitative Local Nanothermometer for Biological Applications.

Femtosecond (fs) laser pulsed excitation of plasmonic nanoparticle (NP)-biomolecule conjugates is a promising method to locally heat biological materials. Studies have demonstrated that fs pulses of light can modulate the activity of DNA or proteins when attached to plasmonic NPs; however, the precision over subsequent biological function remains largely undetermined. Specifically, the temperature the localized biomolecules "experience" remains unknown. We used 55 nm gold nanoparticles (AuNPs) displaying double-stranded (ds) DNA to examine how, for dsDNA with different melting temperatures, the laser pulse energy fluence and bulk solution temperature affect the rate of local DNA denaturation. A universal "template" single-stranded DNA was attached to the AuNP surface, and three dye-labeled probe strands, distinct in length and melting temperature, were hybridized to it creating three individual dsDNA-AuNP bioconjugates. The dye-labeled probe strands were used to quantify the rate and amount of DNA release after a given number of light pulses, which was then correlated to the dsDNA denaturation temperature, resulting in a quantitative nanothermometer. The localized DNA denaturation rate could be modulated by more than threefold over the biologically relevant range of 8-53 °C by varying pulse energy fluence, DNA melting temperature, and surrounding bath temperature. With a modified dissociation equation tailored for this system, a "sensed" temperature parameter was extracted and compared to simulated AuNP temperature profiles. Determining actual biological responses in such systems can allow researchers to design precision nanoscale photothermal heating sources.

Analyzing fidelity and reproducibility of DNA templated plasmonic nanostructures.

Synthetic DNA templated nanostructures offer an excellent platform for the precise spatial and orientational positioning of organic and inorganic nanomaterials. Previous reports have shown its applicability in the organization of plasmonic nanoparticles in a number of geometries for the purpose of realizing tunable nanoscale optical devices. However, translation of nanoparticle-DNA constructs to application requires additional efforts to increase scalability, reproducibility, and formation yields. Understanding all these factors is, in turn, predicated on in-depth analysis of each structure and comparing how formation changes with complexity. Towards the latter goal, we assemble seven unique plasmonic nanostructure symmetries of increasing complexity based on assembly of gold nanorods and nanoparticles on two different DNA origami templates, a DNA triangle and rhombus, and characterize them using gel electrophoresis, atomic force- and transmission electron microscopy, as well as optical spectroscopy. In particular, we focus on how much control can be elicited over yield, reproducibility, shape, size, inter-particle angles, gaps, and plasmon shifts as compared to expectations from computer simulations as structural complexity increases. We discuss how these results can contribute to establishing process principles for creating DNA templated plasmonic nanostructures.