Thermoresponsive fluorescent polymers (TFPs) provide a versatile platform for optical temperature sensing by coupling phase transitions with changes in fluorescence. Here, we report TFPs based on poly(N-isopropylacrylamide) (PNIPAM) and poly(ethylene glycol methacrylate) (PEGMA), incorporating fluorescein acrylate (FluA) as a model fluorophore via reversible addition-fragmentation chain transfer (RAFT) copolymerization. Linear and hyperbranched architectures were synthesized, with different compositions to position cloud point temperatures (Tcp) within a physiologically relevant temperature window, and their fluorescence behavior was investigated as a function of temperature, concentration, and pH. Distinct responses were observed across the Tcp, governed by the interplay between polymer collapse, microenvironmental confinement, and chromophore-chromophore interactions. Systems with low fluorescein loading exhibited fluorescence enhancement near the Tcp followed by quenching at higher temperatures, whereas higher loading systems showed predominantly decreased fluorescence intensity upon heating. Because all samples were compared at equal mass concentration, raw emission intensity scaled primarily with fluorophore loading per chain; we therefore interpret intensity differences cautiously and emphasize the temperature-dependent response as the diagnostic readout. Fluorescence intensity was strongly concentration-dependent, and pH studies revealed changes in both fluorescence intensity and emission maxima, with temperature-responsive fluorescence most prominent at pH 7 and moderately retained at pH 9. Rheological measurements further demonstrated changes in complex viscosity across the Tcp, linking macroscopic behavior to structural transitions. Hyperbranched polymers displayed responses distinct from their linear analogues. We summarize these observations as a set of provisional design rules that link each structural variable, such as composition, fluorophore loading, and architecture, to its dominant effect on Tcp, fluorescence, and viscosity. We note that several of these variables co-vary across the present sample set. Trends are therefore interpreted within controlled compositional windows rather than as fully orthogonal factors, and the resulting rules are intended as practical guidelines for designing polymer-based optical thermometers.

DNA origami is a nanofabrication technique where a long DNA scaffold is folded using locally complementary staple strands to create predesigned two- and three-dimensional structures with desired shapes, sizes, and surface functionalities. While many of these structures have been proposed for applications in biosensing, nanorobotics, and targeted therapeutic delivery, among others, translating this technology from the bench to clinical and industrial settings faces significant challenges, especially in reproducibility, process scalability, and control precision. Microfluidic platforms offer potential solutions to these limitations by providing accurate control, process automation, and easy integration of multiple workflow steps within lab-on-chip devices. This review examines the potential role of microfluidic technologies in DNA origami production, characterization and actuation, highlighting advantages and future directions.

Scaffolded DNA origami has become a valuable nanoscale tool for applications in biomedical and physical sciences. Critical to leveraging the modular and programmable properties of DNA origami nanodevices is access to the scaffold strand, a long single-stranded DNA (ssDNA) of precise length and sequence, which is folded into a compact shape via piecewise base-pairing with many staple strands, short ssDNA oligonucleotides. Current methods to produce and manipulate long ssDNA scaffolds can be costly, time-consuming, and cumbersome. In contrast, methods to produce and manipulate the sequence of double-stranded DNA (dsDNA) are efficient and scalable. Here, we present a method for the rapid isolation of target ssDNA sequences from a variety of dsDNA sources using oligonucleotides as blocking strands that bind continuously to the undesired strand, thereby releasing the target scaffold strand. We report successful ssDNA isolation from linear and supercoiled dsDNAs of various sequences and lengths, ranging from 769 to 15,101 nucleotides. In addition to isolating ssDNA, we demonstrated this approach enables folding of DNA origami directly from dsDNA templates using both blocking and staple strands in a single-pot thermally controlled reaction. Furthermore, we explore multi-scaffold and gene-encoding DNA origami structures, expanding the framework for application-based designs.

The ability to significantly enhance energy transfer processes at the nanoscale requires the simultaneous optimization of molecular-scale orientation and macroscopic photonic enhancement between multiple quantum emitters. However, achieving this dual control has remained a significant experimental challenge, often limited by the stochastic arrangement of emitter assemblies and spatially non-uniform electromagnetic fields in conventional photonic platforms. In this work, we demonstrate a unified architecture that achieves this synergy by combining the structural precision of DNA nanotechnology with the unique field environment generated by epsilon-near-zero (ENZ) materials. Using DNA molecular beacons as programmable emitter scaffolds, we establish fixed donor-acceptor separations and emitter orientations (Atto425/Cy3.5) in two well-defined conformational states: closed hairpin (emitter separation 2 nm) and extended (8.16 nm) configurations. These structures are then embedded in the near-field of a multilayer ENZ metamaterial substrate, which facilitates spatially uniform, enhanced electromagnetic field coupling. Time-resolved photoluminescence measurements demonstrate a significant increase in FRET efficiency for DNA-programmed emitter pairs in the ENZ environment, compared to those on a glass substrate, corresponding to increased donor quenching and shortened donor lifetime. These results establish a scalable experimental pathway for engineering light-matter interactions at molecular scales with applications in next-generation biosensing and quantum photonic technologies.

Packing conformations of molecular aggregates are known to strongly influence the locations and intensities of spectral peaks. Here, we develop the third-order nonlinear spectroscopy signals for a purely electronic model of a molecular dimer, which is a prototype aggregate system. The model-which focuses on excited-state absorption (ESA) pathways in two-dimensional electronic spectra-reveals that orientational averaging leads to diagnostic ESA peak locations for H- and J-dimers. We constructed DNA-templated dimers of cyanine molecules as representative systems and used ultrabroadband two-dimensional electronic spectroscopy measurements to support the predicted signatures arising from the theoretical model. Fitting of steady-state spectra supports the assigned packing conformations. The results elucidate how ESA peaks can be diagnostic spectral signatures of packing conformation. This work lays the foundation for future studies that can include the complicating effects of vibronic states and additional electronic levels.

We demonstrate that coacervates, membraneless organelles formed by liquid-liquid phase separation, sequester and protect short DNA reporters and a functional luciferase gene from enzymatic degradation by various nucleases. Associative coacervates, formed by electrostatic interactions between polyhistidine peptides and ATP, inhibit degradation very efficiently. This protection arises from strong electrostatic interactions between the peptides and oligonucleotides, limiting the enzyme access to recognition and active sites. In contrast, simple coacervates based on a sticker-and-spacer model peptide exhibited limited protection. Oligonucleotide release from associative coacervates can be triggered by external stimuli such as ionic strength or temperature increases, enabling controlled release. Using a cell-free transcription-translation system, we demonstrated that in the presence of nucleases, the associative coacervate samples protected and maintained luciferase production. The ability to protect and controllably release functional genetic material makes coacervates promising candidates for further development as biocompatible delivery vehicles and components of cell-free synthetic biology platforms.

Injectable biomaterials play a vital role in modern medicine, offering tailored functionalities for diverse therapeutic and diagnostic applications. In ophthalmology, for instance, viscoelastic materials are crucial for procedures such as cataract surgery but often leave residues, increasing postoperative risks. This study introduces injectable fluorescent viscoelastics (FluoVs) synthesized via one-step controlled radical copolymerization of oligo(ethylene glycol) acrylate and fluorescein acrylate. These bottlebrush-shaped polymers exhibit enhanced fluorescence intensity for improved traceability and facile removal postsurgery. To prevent aggregation, charged terpolymers were synthesized, ensuring intra- and intermolecular electrostatic repulsion. Dynamic light scattering and energy-conserved dissipative particle dynamics simulations revealed how the fluorescein content and monomer sequence affect the hydrodynamic size of these copolymers. Biocompatibility assessments showed that FluoVs maintained cell viability comparable to commercial hydroxypropyl methylcellulose and nonfluorescent poly(oligo(ethylene glycol) acrylate) controls. The FluoVs combine high fluorescence intensity, low viscosity, and excellent biocompatibility, offering intraoperative traceability and significant advancements for ocular and bioimaging applications.

Cell-free biosensors combine in vitro bacterial transcription-translation systems with operons to detect analytes, such as heavy-metal ions. These sensors are highly desirable due to their easy portability and long shelf life. Typically, the expression of a fluorescent RNA aptamer or protein tied to the presence of an analyte is used as an optical readout for detection in such biosensors. While these readouts have demonstrated tremendous success in testing water potability, the readout is limited by how many different RNA aptamers and proteins can be used simultaneously. The quantum yield of these biological fluorescent molecules is low as well. Recently, we demonstrated a semiconductor quantum dot (QD)-based reporter system that is fully compatible with cell-free transcription-translation systems. Our reporter, abbreviated as QD-PDD (Peptide-PNA DNA Dye), uses nucleic acid specificity to trigger a change in Förster resonance energy transfer (FRET) between the QD and its acceptor fluorophore (Cy3) when a restriction enzyme (BamHI) is expressed. Given the high specificity of nucleic acids and the quantum yield of QDs, the question remained whether QD-PDD reporters could be plugged downstream of heavy-metal cell-free biosensors. Herein, we connected an operon sensitive to cadmium ions to the cell-free expression of BamHI, which triggered a FRET change in the QD-PDD reporter. The operon system can successfully detect cadmium in water-based cadmium chloride solutions. This system serves as a proof of concept showing that QD-PDD can enable the departure of fluorescent biomolecules (aptamers and proteins) in cell-free biosensors.

Abstract Peptide-based liquid-liquid phase separated domains, or coacervates, are a biomaterial gaining new interest due to their exciting potential in fields ranging from biosensing to drug delivery. In this study, we demonstrate that coacervates provide a simple and biocompatible medium to improve nucleic acid biosensors through the sequestration of both the biosensor and target strands within the coacervate, thereby increasing their local concentration. Using the well-established polyarginine (R9) – ATP coacervate system and an energy transfer-based DNA molecular beacon we observed three key improvements: i) a greater than 20-fold reduction of the limit of detection within coacervates when compared to control buffer solutions; ii) an increase in the kinetics, equilibrium was reached more than 4-times faster in coacervates; and iii) enhancement in the dye fluorescent quantum yields within the coacervates, resulting in greater signal-to-noise. The observed benefits translate into coacervates greatly improving bioassay functionality.

Correction for ‘Towards control of excitonic coupling in DNA-templated Cy5 aggregates: the principal role of chemical substituent hydrophobicity and steric interactions’ by Sebastián A. Díaz et al., Nanoscale, 2023, 15, 3284–3299. https://doi.org/10.1039/D2NR05544A.

The number of applications of self-assembled deoxyribonucleic acid (DNA) origami nanoparticles (DNA NPs) has increased drastically, following the development of a variety of single-stranded template DNA (ssDNA) that can serve as the scaffold strand. In addition to viral genomes, such as M13 bacteriophage and lambda DNAs, enzymatically produced ssDNA from various template sources is rapidly gaining traction and being applied as the scaffold for DNA NP preparation. However, separating fully formed DNA NPs that have custom scaffolds from crude assembly mixes is often a multistep process of first separating the ssDNA scaffold from its enzymatic amplification process and then isolating the assembled DNA NPs from excess precursor strands. Only then is the DNA NP sample ready for downstream characterization and application. In this work, we highlight a single-step purification of custom sequence- or M13-derived scaffold-based DNA NPs using photocleavable biotin tethers. The process only requires an inexpensive ultraviolet (UV) lamp, and DNA NPs with up to 90% yield and high purity are obtained. We show the versatility of the process in separating two multihelix bundle structures and a wireframe polyhedral architecture.

Synthetic DNA nanotechnology has emerged as a powerful tool for creating precise nanoscale structures with diverse applications in biotechnology and materials science. Recently, it has evolved to include gene-encoded DNA nanoparticles, which have potentially unique advantages compared to alternative gene delivery platforms. In exciting new developments, we and others have shown how the long single strand within DNA origami nanoparticles, the scaffold strand, can be customized to encode protein-expressing genes and engineer nanoparticles that interface with the transcription-translation machinery for protein production. Remarkably, therefore, DNA nanoparticles - despite their complex three-dimensional shapes - can function as canonical genes. Characteristics such as potentially unlimited gene packing size and low immunogenicity make DNA-based platforms promising for a variety of gene therapy applications. In this review, we first outline various techniques for the isolation of the gene-encoded scaffold strand, a crucial precursor for building protein-expressing DNA nanoparticles. Next, we highlight how features such as sequence design, staple strand optimization, and overall architecture of gene-encoded DNA nanoparticles play a key role in the enhancement of protein expression. Finally, we discuss potential applications of these DNA origami structures to provide a comprehensive overview of the current state of gene-encoded DNA nanoparticles and motivate future directions.

DNA nanotechnology is rapidly gaining traction in numerous applications, each bearing varying degrees of tolerance to the quality and quantity necessary for viable nanostructure function. Despite the distinct objectives of each application, they are united in their reliance on essential analytical techniques, such as purification and characterization. This tutorial aims to guide the reader through the current state of DNA nanotechnology analytical chemistry, outlining important factors to consider when designing, assembling, purifying, and characterizing a DNA nanostructure for downstream applications.

Molecular (dye) aggregates are a materials platform that feature collective excitations, known as excitons, with applications in light harvesting, organic optoelectronics, and nanoscale computing. Several recent works have explored the possibility of using exciton-exciton superpositions as a basic unit in quantum information science (QIS). For their successful use in QIS, it is necessary to maximize both the beating frequency, nu(h), and the decoherence time, tau(d), of the superposition. Although direct measurements of these parameters are challenging, it has been shown that excitonic tau(d) is similar to the decoherence time of the associated optical transitions, that is, optical tau(d). Further, optical tau(d) is related to line broadening, which can readily be measured via absorption spectroscopy. In this work, we characterize line broadening in Cy5 and Cy5.5 monomers and homo- and heterotetramers, tethered to and assembled with Deoxyribonucleic acid (DNA), using steady-state absorption spectroscopy and two-dimensional electronic spectroscopy (2D ES). We also characterize the line broadening of Cy5 free in solution. We find that the width of the primary feature in the tetramer steady-state absorption spectra decreases with increasing Cy5 content. Additionally, the line width of the Cy5 tetramer is smaller than the Cy5 monomer, suggesting that collective excitonic effects may act to reduce the line width. Using 2D ES, we find that homogeneous broadening of all monomers is similar up to time scales of 10 ps; however, after 10 ps DNA appears to cause additional homogeneous broadening, possibly due to large-scale DNA fluctuations. We find using 2D ES that the relative homogeneous and inhomogeneous contributions to spectral broadening in the tetramers are influenced by their composition and excitonic coupling strength. In addition to revealing how aggregate composition influences spectral broadening, this work highlights structural features of dyes that may serve to reduce spectral broadening, which, in turn, may increase optical and excitonic tau(d).

A biohybrid, leaf-spring design of DNA origami functions as a pulsating nanoengine that exploits the DNA-templated RNA transcription mechanism while consuming nucleoside triphosphates as fuel. The nanoengine also drives a nanomechanical follower structure.

We studied the exciton delocalization of indodicarbocyanine 5 dye derivative (Cy5-R) heterodimers templated by a DNA Holliday junction (HJ), which was quantified by the exciton hopping parameter Jm,n. These dyes were modified at the 5 and 5 ' positions of indole rings with substituent (R) H, Cl, tBu, Peg, and hexyloxy (Hex) groups that exhibit different bulkiness and electron-withdrawing/donating capacities. The substituents tune the physical properties of the dyes, such as hydrophobicity (log P) and solvent-accessible surface area (SASA). We tuned the Jm,n of heterodimers by attaching two Cy5-Rs in adjacent and transverse positions along the DNA-HJ. Adjacent heterodimers exhibited smaller Jm,n compared to transverse heterodimers, and some adjacent heterodimers displayed a mixture of H- and J-like aggregates. Most heterodimers exhibited Jm,n values within the ranges of the corresponding homodimers, but some heterodimers displayed synergistic exciton delocalization that resulted in larger Jm,n compared to their homodimers. We then investigated how chemically distinct Cy5-R conjugated to DNA can interact to create delocalized excitons. We determined that heterodimers involving Cy5-H and Cy5-Cl and a dye with larger substituents (bulky substituents and large SASA) such as Cy5-Peg, Cy5-Hex, and Cy5-tBu resulted in larger Jm,n. The combination provides steric hindrance that optimizes co-facial packing (bulky Cy5-R) with a smaller footprint (small SASA) that maximizes proximity. The results of this study lay a groundwork for rationally optimizing the exciton delocalization in dye aggregates for developing next-generation technologies based on optimized exciton transfer efficiency such as quantum information systems and biomedicine. We studied the exciton delocalization of indodicarbocyanine 5 dye derivative (Cy5-R) heterodimers templated by a DNA Holliday junction (HJ), which was quantified by the exciton hopping parameter Jm,n.

An attractive strategy to improve the energy transfer properties of synthetic dye networks is to optimize the excitonic coupling between the dyes to increase the energy transfer rates. To explore this possibility, we investigated the use of J-like cyanine dye dimers (Cy3 and Cy5 dimers) on DNA duplexes as energy transfer relays in molecular photonic wires. This approach is based on the use of the collective emission dipole of a J-dimer to enhance the FRET rate between the dimer relay and a remote acceptor dye. Experimentally, we find that in room temperature aqueous buffer conditions, the dimer relay provided no benefit in the energy transfer quantum yield relative to a simple monomer relay. Further investigation led us to determine that enhanced nonradiative relaxation, non-ideal dye orientation within the dimer, and unfavorable dye orientation between the dimer and the acceptor dye limit energy transfer through the dimer relay. We hypothesized that nonradiative relaxation was the largest factor and demonstrated this by placing the sample in a viscous solvent or by cooling the sample, which dramatically improved the energy transfer through the J-like dimer relay. Similar to how the formation of DNA-templated J-like dimers has improved, the practical use of J-like dimers to optimize energy transfer quantum efficiency will require improvements in the ability to control the orientation between dyes to reach its full potential.

DNA nanotechnology has made initial progress toward developing gene-encoded DNA origami nanoparticles (NPs) that display potential utility for future gene therapy applications. However, due to the challenges involved with gene delivery into cells including transport through the membrane, intracellular targeting, and inherent expression of nucleases along with interference from other active proteins, it can be difficult to more directly study the effect of DNA NP design on subsequent gene expression. In this work, we demonstrate an approach for studying the expression of gene-encoding DNA origami NPs without the use of cells. We utilize a pure E. coli-derived cell-free transcription-translation (TXTL) system, which is composed of optimized components from bacterial expression, for benchtop studies to assess how the promoter sequence in conjunction with structural design of the DNA NP template affects gene expression. The gene for an optimized Renilla luciferase was first amplified into a single-stranded (ss) scaffold strand and then folded into a 12-helix bundle DNA NP with exogenous staple strands as a test platform. Using luciferase-based bioluminescence assays to characterize the relative protein expression level, it was found that the gene can still be transcribed when folded, albeit at a lower rate than the double-stranded DNA gene segment. On comparing three variants of DNA NP with different promoter configurations, results indicate that a promoter designed to remain in ssDNA form has reduced protein expression from the DNA NP, and replacing the promoter sequence with an arbitrary sequence significantly lowers protein expression. This work demonstrates the power inherent in cell-free TXTL systems as an aid to study the gene expression capabilities of DNA NPs toward design and development of future applications.

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 AlexaFluor (TM) 647 (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.