Semiconductor quantum dots (QD) combine bright and color-tunable photoluminescence (PL) with large surfaces. Thus, merging multiplexed PL detection and specific biorecognition on a single QD is in principle possible. By attaching multiple DNAs per QD and adjusting terbium (Tb) to QD Förster resonance energy transfer (FRET) with sub-nanometer resolution, we designed dual-color FRET-nanoprobes with six distinguishable signals. The hexaplexing molecular probes combined two PL colors with three PL decays while requiring only a single excitation wavelength for rapid and simple biosensing. Implementation into a sensitive diagnostic assay was demonstrated via the specific quantification of six different DNA targets at picomolar concentrations (2 to 40 femtomole) from a single 200 µL sample. The orthogonality of specific biorecognition and FRET multiplexing on the nanosurface makes the QD-based FRET probes extendable to the sensitive quantification of potentially any biomarker or biological interaction at even higher multiplexing capability. Our results demonstrate that luminescent nanoprobes are capable of translating sophisticated multiplexing approaches into relatively simple biosensing methods.

An intensity level-based method for analyzing the emission of single quantum dots and pairs of quantum dots from wide-field EMCCD camera images and simultaneously determining their lifetimes using a wide-field time-resolved nocturnal camera (NCam) is discussed. Simulated data is generated by accounting for the probability to image photons from two quantum dots, as well as background emission and dark counts on the pixels of the two detectors. Segregating data based on different intensity levels enables us to localize the emitters with <10-nm localization precision, measure the interparticle distances within <2-nm, and determine the photoluminescence decay histogram of both emitters. The technique is demonstrated using simulated photon emission data and super resolution imaging from a pair of blinking dual-emitters with variable intensity ratios and spatial separations. This method will be applied to image pairs of quantum dots patterned on DNA origami in close enough proximity to undergo resonance energy transfer.

Biocatalytic production of human milk oligosaccharides (HMOs) depends on efficient synthesis of the sugar backbone core. Previous studies engineered a D746E variant of a GH20 β-N-acetylhexosaminidase (BbhI) that appears promising for industrial synthesis of the trisaccharide HMO core precursor lacto-N-triose II (LNTII) using a chemically prepared N-acetylglucosamine oxazoline (NAG-oxa). In this study, a NMR-based quantitative assay was developed to explore the Michaelis-Menten kinetics of N-acetylated sugar oxazoline consumption by D746E BbhI. These results show that chemical conversion of N-acetylhexosamine to an oxazoline-activated sugar dramatically increases turnover relative to p-nitrophenylated sugar (kcat = 277 ± 9 s-1 for NAG-oxa consumption). Enzyme-catalyzed consumption was also observed for N-acetylgalactosamine and N-acetylmannosamine oxazolines. These results represent the first reported Michaelis-Menten kinetics of a glycosynthase with respect to sugar oxazoline consumption and suggest that utilization of substrates beyond that of standard p-nitrophenylated sugars may be useful in carbohydrate-active enzyme (CAZyme) engineering and broaden product oligosaccharide diversity.

Abstract The assembly of molecular photonic wires (MPWs) on DNA scaffolds offers a powerful platform for controlling nanoscale energy transfer. This work demonstrates how the geometric configuration of an excitonic relay, composed of a cyanine (Cy5) dye dimer, regulates energy flow within an MPW. Exploiting linker chemistry, either H-type or J-type aggregates are selectively formed at room temperature. H-type dimers act as energy transfer inhibitors, while J-type dimers function as effective energy relays. In an optimized architecture, J-dimer MPWs outperform equivalent systems using monomeric relays. This performance is significantly amplified upon transitioning the system from solution to solid-state films, where the energy transfer efficiency of J-dimer wires is enhanced by up to 300% relative to monomeric versions. Experimental results also support approximating the dimers as single-point dipoles for Förster resonance energy transfer considerations. These results establish a robust strategy for engineering the optical properties of molecular materials, where nanoscale energy transport is precisely directed by controlling the geometry of excitonic aggregates.

Exposure to organophosphates poses a threat to aquatic ecosystems and the public due to their acute toxicity. The dangers caused by organophosphates motivate the development of technologies for prompt detection. Therefore, engineering a portable and compact device that can monitor organophosphate vapors is vital. Electrochemical biosensors present a unique solution for rapid and selective detection of organophosphates, as these sensors are small and sense in real-time. Herein, this study investigates a dual-modal electrochemical system for the detection of dimethyl methylphosphonate (DMMP), a model organophosphate. The enzymes phosphotriesterase (PTE) and acetylcholinesterase (AChE) were chosen for detecting DMMP vapors, as these enzymes chemically engage with the target substrate and produce electrochemically measurable signals. These reaction-specific signals can be read using open circuit potentiometry and amperometry, which produce a turn-on/turn-off signal readout. The turn-on/turn-off signal readout demonstrates the dual-modal mechanism and serves as a complementary verification, thus circumventing the propensity for false positives typically associated with AChE-functionalized biosensors. Deep eutectic solvent films (∼20 μL) compatible with each enzyme were developed to achieve a truly field-deployable device that does not dry out during operation. The PTE- and AChE-modified biosensors yielded detection limits of 17.6 ± 8.7 and 2.5 ± 0.6 ppm, respectively. The dual-sensing device was further validated against a broader range of organophosphates, demonstrating the device's utility as a functional organophosphate sensor. A dual-modal biosensing system presents a unique approach for organophosphate vapor mapping, which results in proper agricultural stewardship.

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.

CRISPR-Cas systems have revolutionized molecular diagnostics through their specificity and programmability, yet their broad adoption is hindered by the reliance on expensive and complex instrumentation. Here, we present an optimized quantum dot (QD) molecular beacon (QD-MB) platform that integrates Förster resonance energy transfer (FRET)-based detection with CRISPR-Cas functionality, achieving sub-picomolar sensitivity without the need for target amplification. By systematically tuning components, including His-tag modifications for improved QD conjugation, nucleic acid hairpin structures for enhanced enzyme interaction, and QD surface passivation strategies, we demonstrate a two-order-of-magnitude improvement in detection sensitivity. Using LwaCas13a and RNA targets, the limit of detection (LOD) decreased to under 1 pM with plate-reader-based fluorescence measurements and below 10 pM with a lamp-and-smartphone setup, establishing the feasibility of portable, field-ready applications. This work highlights the transformative potential of QD-MBs in biosensing and sets a foundation for further advances in CRISPR-based diagnostics and nanotechnology-enabled sensing platforms.

Semiconductor nanoplatelets possess exceptional optical properties that make them promising candidates for next-generation optoelectronic applications. However, unlike quantum dots where absorption spectroscopy alone can determine both size and concentration, nanoplatelets present a significant characterization challenge: the absorption peak position reveals only thickness, providing no information about lateral dimensions or concentration. This limitation forces researchers to rely on time-consuming elemental analysis techniques for complete sample characterization. Here, we present an experimentally verified theoretical framework that predicts the frequency-dependent absorption coefficient of randomly oriented CdSe, CdS, and CdTe nanoplatelets, enabling concentration determination from absorption measurements and lateral size estimates. Our model shows that the integrated absorption coefficient depends universally on nanoplatelet surface area and thickness, yielding a practical tool to extract concentrations without laborious elemental analysis. This approach bridges the characterization gap between quantum dots and nanoplatelets, offering a streamlined method for sample analysis that could accelerate nanoplatelet research and applications.

We describe the facile synthesis of three different classes of nanoparticle (NP) surface ligands that are particularly useful to prepare hydrophilic, biocompatible semiconductor quantum dots (QDs) and gold nanoparticles (AuNPs). The ligands consist of a bidentate thioctic acid (TA) or dihydrolipoic acid anchor group, allowing for high-affinity NP attachment, and are synthesized in a simple manner without compromising the hydrophilicity and utility for further applications. First, we applied a trifluoroacetamide protecting group to amine-terminated TA-based ligands to suppress irreversible gelation, which is common with amine-appended TA-based ligands. Both the protection and deprotection steps were straightforward, and the successful in situ deprotection was demonstrated by gel electrophoresis and dye conjugation assays. Second, our TA-based zwitterionic ligands with a bis(carboxyethyl)amino group were integrated with oligo(ethylene glycol) or poly(ethylene glycol) (PEG) groups to enhance the biocompatibility. Their antifouling properties were examined by a gel electrophoresis assay with bovine serum albumin. Lastly, hydroxy-terminated branched TA-based ligands were synthesized using a minimum number of steps as alternatives to hydroxy-terminated TA-based PEG ligands, which require laborious purification steps. The utility of the compact hydroxy-terminated ligands was further demonstrated by (i) gel electrophoresis assays to explore their antifouling properties and (ii) photoluminescence wavelength tuning during the direct aqueous synthesis of luminescent gold nanoclusters. Inherent benefits of the ligand design were demonstrated beyond QDs as AuNPs functionalized with the same compact ligand series showed similar colloidal properties. The simple designs along with a variety of proven utilities highlight the strong potential of these ligands to expand NP capabilities in many biological applications.

Förster resonance energy transfer (FRET) spectroscopy and microscopy are constantly expanding sensing techniques for analyzing biomolecular interactions. In addition to the biological recognition molecules and biological or chemical analytes, the most important components for designing FRET biosensing systems are the materials that constitute the FRET donor-acceptor pair. These FRET materials consist of small molecules, biological or chemical nanoscaffolds, or nanomaterials that function in the ultraviolet, visible, or infrared spectral range. They can absorb light, fluoresce or phosphoresce with lifetimes ranging from picoseconds to milliseconds, and can be applied for sensing in situ, in vitro, and in vivo. Organic dyes and quenchers, fluorescent or light harvesting proteins, or quantum dots are only some examples from the ever growing FRET material toolbox. A particular example are gold nanoparticles, whose strong localized surface plasmon resonance makes them frequently used as nanosurface energy transfer (NSET) acceptors. After a short recapitulation of FRET and NSET theory, we review a wide variety of FRET and NSET materials, provide representative examples of FRET/NSET systems and applications for each material, and critically discuss the benefits and drawbacks of their properties.

Allowing coupled enzymes to crosslink with nanoparticles (NPs) into nanoclusters has been shown to facilitate them engaging in the most efficient form of multienzymatic catalysis, namely that of intermediary channeling. Utilizing a previously validated nanoparticle-scaffolded seven enzyme cascade from glycolysis that processes glucose into 3-phosphoglycerate, we begin by confirming that non-cadmium containing ZnSe/ZnS core/shell quantum dots (QDs) made from non-toxic and earth abundant materials can replace Cd-containing QDs as a scaffolding material in the multienzyme clusters while still providing access to improved channeling activity. We then investigate the role of enzyme assembly order within mixed NP systems that consist of both spherical QDs and rectangular 2-dimensional nanoplatelets (NPLs). Along with physicochemical confirmation of enzyme assembly to the QDs and enzyme-induced cluster formation, the rate of overall catalytic flux for each of the systems was monitored under different assembly conditions. The results reveal that adjusting relative NP concentration normalized to surface area, enzyme assembly order, and choice of initial material in any mixed NP clustered configuration are critical to attaining further improvements in catalytic flux via channeling. The potential ramifications of these observations in the context of assembling designer biosynthetic cascades that use bulk feedstock materials derived from agriculture to create new and useful products are then discussed.Graphical AbstractSchematic of a self-assembled mixed QD-NPL-enzyme system engaged in 7-enzyme sequential substrate channeling.

In recent years, DNA scaffolds have been utilized to organize dye molecules into aggregates with tailored optical and photophysical properties. While dye separation can be controlled with nanometer-scale accuracy, controlling the relative dye orientation in an aggregate on DNA remains challenging. In this work, we investigate varying the length of the two-point linker between indodicarbocyanine (Cy5) dyes and the DNA template as a method to better control the resulting dimer geometry. To test this approach, we synthesize Cy5 with either 2-carbon or 4-carbon two-point linkers and compare their behavior to commercially available Cy5 with 3-carbon two-point linkers. Using experimental spectroscopy, theoretical modeling, and molecular dynamics simulations, we demonstrate that shortening the linker from 3-carbon to 2-carbon limits the π-π interactions between dyes, thereby promoting the formation of J-like Cy5 dimers. Conversely, increasing the linker length provides the dye more freedom of motion, allowing greater π-π interactions and yielding dimers with greater H-like character. Furthermore, shorter linkers can restrict dye accessible volume, which, under the driving force of π-π interactions, suppresses heterogeneity in dye packing for specific placements of Cy5 on double stranded DNA and DNA Holliday junction scaffolds. These results emphasize the importance of dye linker chemistry in determining important optical and photophysical properties of DNA-scaffolded dye aggregates. They also suggest that tuning the length of the dye linker is an effective strategy to overcome two challenges that currently limit DNA-scaffolded dye aggregates in photonics applications: gaining control of dye aggregate geometry and suppressing heterogeneity in dye packing.

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.

Co-assembling enzymes with nanoparticles (NPs) into nanoclusters allows them to access channeling, a highly efficient form of multienzyme catalysis. Using pyruvate kinase (PykA) and lactate dehydrogenase (LDH) to convert phosphoenolpyruvic acid to lactic acid with semiconductor quantum dots (QDs) confirms how enzyme cluster formation dictates the rate of coupled catalytic flux (kflux) across a series of differentially sized/shaped QDs and 2D nanoplatelets (NPLs). Enzyme kinetics and coupled flux were used to demonstrate that by mixing different NP systems into clusters, a >10× improvement in kflux is observed relative to free enzymes, which is also ≥2× greater than enhancement on individual NPs. Cluster formation was characterized with gel electrophoresis and transmission electron microscopy (TEM) imaging. The generalizability of this mixed-NP approach to improving flux is confirmed by application to a seven-enzyme system. This represents a powerful approach for accessing channeling with almost any choice of enzymes constituting a multienzyme cascade.