Multivalent Display of Erythropoietin on Quantum Dots Enhances Aquaporin-4 Expression and Water Transport in Human Astrocytes In Vitro.

In mammalian cells, growth factor-induced intracellular signaling and protein synthesis play a critical role in cellular physiology and homeostasis. In the brain's glymphatic system (GS), the water-conducting activity of aquaporin-4 (AQPN-4) membrane channels (expressed in polarized fashion on astrocyte end-feet) mediates the clearance of wastes through the convective transport of fluid and solutes through the perivascular space. The glycoprotein erythropoietin (EPO) has been shown to induce the astrocyte expression of AQPN-4 via signaling through the EPO receptor and the JAK/STAT signaling pathway. Here, we self-assemble EPO in a multivalent fashion onto the surface of semiconductor quantum dots (QDs) (driven by polyhistidine-based self-assembly) to drive the interaction of the bioconjugates with EPOR on human astrocytes (HA). This results in a 2-fold augmentation of JAK/STAT signaling activity and a 1.8-fold enhancement in the expression of AQPN-4 in cultured primary HA compared to free EPO. This translates into a 2-fold increase in the water transport rate in HA cells as measured by the calcein AM water transport assay. Importantly, EPO-QD-induced augmented AQPN-4 expression does not elicit any deleterious effect on the astrocyte viability. We discuss our results in the context of the implications of EPO-nanoparticle (NP) bioconjugates for use as research tools to understand the GS and their potential as therapeutics for the modulation of GS function. More generally, our results illustrate the utility of NP bioconjugates for the controlled modulation of growth factor-induced intracellular signaling.

The Role of Ligands in the Chemical Synthesis and Applications of Inorganic Nanoparticles.

The design of nanoparticles is critical for their efficient use in many applications ranging from biomedicine to sensing and energy. While shape and size are responsible for the properties of the inorganic nanoparticle core, the choice of ligands is of utmost importance for the colloidal stability and function of the nanoparticles. Moreover, the selection of ligands employed in nanoparticle synthesis can determine their final size and shape. Ligands added after nanoparticle synthesis infer both new properties as well as provide enhanced colloidal stability. In this article, we provide a comprehensive review on the role of the ligands with respect to the nanoparticle morphology, stability, and function. We analyze the interaction of nanoparticle surface and ligands with different chemical groups, the types of bonding, the final dispersibility of ligand-coated nanoparticles in complex media, their reactivity, and their performance in biomedicine, photodetectors, photovoltaic devices, light-emitting devices, sensors, memory devices, thermoelectric applications, and catalysis.

The Rare Bone Disease TeleECHO Program: Leveraging Telehealth to Improve Rare Bone Disease Care.

PURPOSE OF REVIEW: Rare bone diseases constitute ~ 5% of all known rare diseases and can require complex, multidisciplinary care. Advancing access to current medical knowledge is an important strategy for improving care for rare bone diseases throughout the world. To support this goal, the Rare Bone Disease Alliance launched the Rare Bone Disease TeleECHO in 2019. RECENT FINDINGS: The Rare Bone Disease TeleECHO is a monthly video teleconference that fosters a collegial community of practice and opportunities for active learning through interactive case-based learning. TeleECHO relies on a hub-and-spoke model, where medical professionals at the "hub" provide support and expertise for other healthcare providers, or the "spokes". Evidence of the global reach of the program as well as qualitative feedback from registrants supports the need for rare bone disease education and the value of the TeleECHO model. The Rare Bone Disease TeleECHO helps meet the challenge of disseminating rapidly expanding rare bone disease knowledge by leveraging telehealth.

Transducing Protease Activity into DNA Output for Developing Smart Bionanosensors.

DNA can process information through sequence-based reorganization but cannot typically receive input information from most biological processes and translate that into DNA compatible language. Coupling DNA to a substrate responsive to biological events can address this limitation. A two-component sensor incorporating a chimeric peptide-DNA substrate is evaluated here as a protease-to-DNA signal convertor which transduces protease activity through DNA gates that discriminate between different input proteases. Acceptor dye-labeled peptide-DNAs are assembled onto semiconductor quantum dot (QD) donors as the input gate. Addition of trypsin or chymotrypsin cleaves their cognate peptide sequence altering the efficiency of Förster resonance energy transfer (FRET) with the QD and frees a DNA output which interacts with a tetrahedral output gate. Downstream output gate rearrangement results in FRET sensitization of a new acceptor dye. Following characterization of component assembly and optimization of individual steps, sensor ability to discriminate between the two proteases is confirmed along with effects from joint interactions where potential for cross-talk is highest. Processing multiple bits of information for a sensing outcome provides more confidence than relying on a single change especially for the discrimination between different targets. Coupling other substrates to DNA that respond similarly could help target other types of enzymes.

Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications.

Luminescent semiconductor quantum dots (QDs) are one of the more popular nanomaterials currently utilized within biological applications. However, what is not widely appreciated is their growing role as versatile energy transfer (ET) donors and acceptors within a similar biological context. The progress made on integrating QDs and ET in biological configurations and applications is reviewed in detail here. The goal is to provide the reader with (1) an appreciation for what QDs are capable of in this context, (2) how this field has grown over a relatively short time span, and, in particular, (3) how QDs are steadily revolutionizing the development of new biosensors along with a myriad of other photonically active nanomaterial-based bioconjugates. An initial discussion of QD materials along with key concepts surrounding their preparation and bioconjugation is provided given the defining role these aspects play in the QDs ability to succeed in subsequent ET applications. The discussion is then divided around the specific roles that QDs provide as either Förster resonance energy transfer (FRET) or charge/electron transfer donor and/or acceptor. For each QD-ET mechanism, a working explanation of the appropriate background theory and formalism is articulated before examining their biosensing and related ET utility. Other configurations such as incorporation of QDs into multistep ET processes or use of initial chemical and bioluminescent excitation are treated similarly. ET processes that are still not fully understood such as QD interactions with gold and other metal nanoparticles along with carbon allotropes are also covered. Given their maturity, some specific applications ranging from in vitro sensing assays to cellular imaging are separated and discussed in more detail. Finally a perspective on how this field will continue to evolve is provided.

Concurrent Modulation of Quantum Dot Photoluminescence Using a Combination of Charge Transfer and Förster Resonance Energy Transfer: Competitive Quenching and Multiplexed Biosensing Modality.

An emerging trend with semiconductor quantum dots (QDs) is their use as scaffolds to assemble multiple energy transfer pathways. Examples to date have combined various competitive and sequential Förster resonance energy transfer (FRET) pathways between QDs and fluorescent dyes, luminescent lanthanide complexes, and bioluminescent proteins. Here, we show that the photoluminescence (PL) of QD bioconjugates can also be modulated by a combination of FRET and charge transfer (CT), and characterize the concurrent effects of these mechanistically different pathways using PL measurements at both the ensemble and the single particle level. Peptides were distally labeled with either a fluorescent dye that quenched QD PL through FRET or a ruthenium(II) phenanthroline complex that quenched QD PL through electron transfer. The labeled peptides were assembled around a central CdSe/ZnS QD at different ratios, tuning the relative rates of FRET and CT, which were competitive quenching pathways. The concurrent effects of FRET and CT were predictable from a rate analysis that was calibrated to the isolated effects of each of these pathways. Notably, the dye/QD PL intensity ratio reflected changes in the relative rate of FRET but was approximately independent of CT. In turn, the sum of the QD and dye PL intensities, when adjusted for quantum yields, reflected changes in the relative rate of CT quenching, approximately independent of FRET. The capacity for multiplexed sensing of protease activity was demonstrated using these two orthogonal detection channels. Combined CT-FRET configurations with QDs are thus promising for applications in bioanalysis, sensing, and imaging, and may prove useful in other photonic applications.

Electric Field Modulation of Semiconductor Quantum Dot Photoluminescence: Insights Into the Design of Robust Voltage-Sensitive Cellular Imaging Probes.

The intrinsic properties of quantum dots (QDs) and the growing ability to interface them controllably with living cells has far-reaching potential applications in probing cellular processes such as membrane action potential. We demonstrate that an electric field typical of those found in neuronal membranes results in suppression of the QD photoluminescence (PL) and, for the first time, that QD PL is able to track the action potential profile of a firing neuron with millisecond time resolution. This effect is shown to be connected with electric-field-driven QD ionization and consequent QD PL quenching, in contradiction with conventional wisdom that suppression of the QD PL is attributable to the quantum confined Stark effect.

Combining semiconductor quantum dots and bioscaffolds into nanoscale energy transfer devices.

Significant advances have been made in the development of nanoscale devices capable of exciton transport via Förster resonance energy transfer. Several requirements must be met for effective operation, including a reliable energy-harvesting source along with highly organized, precisely placed energy relay elements. For the latter, biological scaffolds such as DNA provide a customizable, symmetric, and stable structure that can be site-specifically modified with organic fluorophores. Here, advancements in nanoscale energy transfer devices incorporating semiconductor nanocrystals and bioscaffolds are reviewed with discussion of biofunctionalization, linker chemistries, design considerations, and concluding with applications in light harvesting, multiplexed biosensing, and optical logic.

3-Dimensional Tracking of Non-blinking 'Giant' Quantum Dots in Live Cells.

While semiconductor quantum dots (QDs) have been used successfully in numerous single particle tracking (SPT) studies due to their high photoluminescence efficiency, photostability, and broad palette of emission colors, conventional QDs exhibit fluorescence intermittency or 'blinking,' which causes ambiguity in particle trajectory analysis and limits tracking duration. Here, non-blinking 'giant' quantum dots (gQDs) are exploited to study IgE-FcεRI receptor dynamics in live cells using a confocal-based 3D SPT microscope. There is a 7-fold increase in the probability of observing IgE-FcεRI for longer than 1 min using the gQDs compared to commercially available QDs. A time-gated photon-pair correlation analysis is implemented to verify that selected SPT trajectories are definitively from individual gQDs and not aggregates. The increase in tracking duration for the gQDs allows the observation of multiple changes in diffusion rates of individual IgE-FcεRI receptors occurring on long (>1 min) time scales, which are quantified using a time-dependent diffusion coefficient and hidden Markov modeling. Non-blinking gQDs should become an important tool in future live cell 2D and 3D SPT studies, especially in cases where changes in cellular dynamics are occurring on the time scale of several minutes.

Optimizing the conversion of phosphoenolpyruvate to lactate by enzymatic channeling with mixed nanoparticle display.

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.

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.

Optimizing protein coordination to quantum dots with designer peptidyl linkers.

Semiconductor quantum dots (QDs) demonstrate select optical properties that make them of particular use in biological imaging and biosensing. Controlled attachment of biomolecules such as proteins to the QD surface is thus critically necessary for development of these functional nanobiomaterials. QD surface coatings such as poly(ethylene glycol) impart colloidal stability to the QDs, making them usable in physiological environments, but can impede attachment of proteins due to steric interactions. While this problem is being partially addressed through the development of more compact QD ligands, here we present an alternative and complementary approach to this issue by engineering rigid peptidyl linkers that can be appended onto almost all expressed proteins. The linkers are specifically designed to extend a terminal polyhistidine sequence out from the globular protein structure and penetrate the QD ligand coating to enhance binding by metal-affinity driven coordination. α-Helical linkers of two lengths terminating in either a single or triple hexahistidine motif were fused onto a single-domain antibody; these were then self-assembled onto QDs to create a model immunosensor system targeted against the biothreat agent ricin. We utilized this system to systematically evaluate the peptidyl linker design in functional assays using QDs stabilized with four different types of coating ligands including poly(ethylene glycol). We show that increased linker length, but surprisingly not added histidines, can improve protein to QD attachment and sensor performance despite the surface ligand size with both custom and commercial QD preparations. Implications for these findings on the development of QD-based biosensors are discussed.

Evaluation of diverse peptidyl motifs for cellular delivery of semiconductor quantum dots.

Cell-penetrating peptides (CPPs) have rapidly become a mainstay technology for facilitating the delivery of a wide variety of nanomaterials to cells and tissues. Currently, the library of CPPs to choose from is still limited, with the HIV TAT-derived motif still being the most used. Among the many materials routinely delivered by CPPs, nanoparticles are of particular interest for a plethora of labeling, imaging, sensing, diagnostic, and therapeutic applications. The development of nanoparticle-based technologies for many of these uses will require access to a much larger number of functional peptide motifs that can both facilitate cellular delivery of different types of nanoparticles to cells and be used interchangeably in the presence of other peptides and proteins on the same surface. Here, we evaluate the utility of four peptidyl motifs for their ability to facilitate delivery of luminescent semiconductor quantum dots (QDs) in a model cell culture system. We find that an LAH4 motif, derived from a membrane-inserting antimicrobial peptide, and a chimeric sequence that combines a sweet arrow peptide with a portion originating from the superoxide dismutase enzyme provide effective cellular delivery of QDs. Interestingly, a derivative of the latter sequence lacking just a methyl group was found to be quite inefficient, suggesting that even small changes can have significant functional outcomes. Delivery was effected using 1 h incubation with cells, and fluorescent counterstaining strongly suggests an endosomal uptake process that requires a critical minimum number or ratio of peptides to be displayed on the QD surface. Concomitant cytoviability testing showed that the QD-peptide conjugates are minimally cytotoxic in the model COS-1 cell line tested. Potential applications of these peptides in the context of cellular delivery of nanoparticles and a variety of other (bio)molecules are discussed.

Proteolytic activity at quantum dot-conjugates: kinetic analysis reveals enhanced enzyme activity and localized interfacial "hopping".

Recent studies show that polyvalent, ligand-modified nanoparticles provide significantly enhanced binding characteristics compared to isolated ligands. Here, we assess the ability of substrate-modified nanoparticles to provide enhanced enzymatic activity. Energy transfer assays allowed quantitative, real-time measurement of proteolytic digestion at polyvalent quantum dot-peptide conjugates. Enzymatic progress curves were analyzed using an integrated Michaelis-Menten (MM) formalism, revealing mechanistic details, including deviations from classic MM-behavior. A "hopping" mode of proteolysis at the nanoparticle was identified, confirming enhanced activity.

Self assembling nanoparticle enzyme clusters provide access to substrate channeling in multienzymatic cascades.

Access to efficient enzymatic channeling is desired for improving all manner of designer biocatalysis. We demonstrate that enzymes constituting a multistep cascade can self-assemble with nanoparticle scaffolds into nanoclusters that access substrate channeling and improve catalytic flux by orders of magnitude. Utilizing saccharification and glycolytic enzymes with quantum dots (QDs) as a model system, nanoclustered-cascades incorporating from 4 to 10 enzymatic steps are prototyped. Along with confirming channeling using classical experiments, its efficiency is enhanced several fold more by optimizing enzymatic stoichiometry with numerical simulations, switching from spherical QDs to 2-D planar nanoplatelets, and by ordering the enzyme assembly. Detailed analyses characterize assembly formation and clarify structure-function properties. For extended cascades with unfavorable kinetics, channeled activity is maintained by splitting at a critical step, purifying end-product from the upstream sub-cascade, and feeding it as a concentrated substrate to the downstream sub-cascade. Generalized applicability is verified by extending to assemblies incorporating other hard and soft nanoparticles. Such self-assembled biocatalytic nanoclusters offer many benefits towards enabling minimalist cell-free synthetic biology.

Quantum dots as simultaneous acceptors and donors in time-gated Förster resonance energy transfer relays: characterization and biosensing.

The unique photophysical properties of semiconductor quantum dot (QD) bioconjugates offer many advantages for active sensing, imaging, and optical diagnostics. In particular, QDs have been widely adopted as either donors or acceptors in Förster resonance energy transfer (FRET)-based assays and biosensors. Here, we expand their utility by demonstrating that QDs can function in a simultaneous role as acceptors and donors within time-gated FRET relays. To achieve this configuration, the QD was used as a central nanoplatform and coassembled with peptides or oligonucleotides that were labeled with either a long lifetime luminescent terbium(III) complex (Tb) or a fluorescent dye, Alexa Fluor 647 (A647). Within the FRET relay, the QD served as a critical intermediary where (1) an excited-state Tb donor transferred energy to the ground-state QD following a suitable microsecond delay and (2) the QD subsequently transferred that energy to an A647 acceptor. A detailed photophysical analysis was undertaken for each step of the FRET relay. The assembly of increasing ratios of Tb/QD was found to linearly increase the magnitude of the FRET-sensitized time-gated QD photoluminescence intensity. Importantly, the Tb was found to sensitize the subsequent QD-A647 donor-acceptor FRET pair without significantly affecting the intrinsic energy transfer efficiency within the second step in the relay. The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event. Potential benefits of this time-gated FRET approach include: eliminating background fluorescence, accessing two approximately independent FRET mechanisms in a single QD-bioconjugate, and multiplexed biosensing based on spectrotemporal resolution of QD-FRET without requiring multiple colors of QD.

Multiplexed tracking of protease activity using a single color of quantum dot vector and a time-gated Förster resonance energy transfer relay.

Semiconductor quantum dots (QDs) are attractive probes for optical sensing and imaging due to their unique photophysical attributes and nanoscale size. In particular, the development of assays and biosensors based on QDs and Förster resonance energy transfer (FRET) continues to be a prominent focus of research. Here, we demonstrate the application of QDs as simultaneous donors and acceptors in a time-gated FRET relay for the multiplexed detection of protease activity. In contrast to the current state-of-the-art, which uses multiple colors of QDs, multiplexing was achieved using only a single color of QD. The other constituents of the FRET relay, a luminescent terbium complex and fluorescent dye, were assembled to QDs via peptides that were selected as substrates for the model proteases trypsin and chymotrypsin. Loss of prompt FRET between the QD and dye signaled the activity of chymotrypsin; loss of time-gated FRET between the terbium and QD signaled the activity of trypsin. We applied the FRET relay in a series of quantitative, real-time kinetic assays of increasing biochemical complexity, including multiplexed sensing, measuring inhibition in a multiplexed format, and tracking the proteolytic activation of an inactive pro-protease to its active form in a coupled, multienzyme system. These capabilities were derived from a ratiometric analysis of the two FRET pathways in the relay and permitted extraction of initial reaction rates, enzyme specificity constants, and apparent inhibition constants. This work adds to the growing body of research on multifunctional nanoparticles and introduces multiplexed sensing as a novel capability for a single nanoparticle vector. Furthermore, the ability to track both enzymes within a coupled biological system using one vector represents a significant advancement for nanoparticle-based biosensing. Prospective applications in biochemical research, applied diagnostics, and drug discovery are discussed.

Quantum dots and fluorescent protein FRET-based biosensors.

There has been considerable recent interest in the creation of nanoparticle-biomolecule hybrid materials for uses such as in vitro and in vivo biosensing, biological imaging, and drug -delivery. Nanoparticles have a high surface to volume ratio, making them capable of being decorated with -various biomolecules on their surface which retain their biological activity. Techniques to bind these biomolecules to nanoparticle surfaces are also advancing rapidly. Here we demonstrate hybrid materials assembled around CdSe/ZnS core/shell semiconductor quantum dots (QDs). These intrinsically fluorescent materials are conjugated to the fluorescent proteins YFP, mCherry and the light harvesting complex b-phycoerythrin (b-PE). QDs have fluorescent properties that make them ideal as donor fluorophores for Förster resonance energy transfer (FRET) while the fluorescent proteins are able to act as FRET acceptors displaying many advantages over organic dyes. We examine FRET interactions between QDs and all three fluorescent proteins. Furthermore, we show QD-mCherry hybrid materials can be utilized for in vitro biosensing of caspase-3 enzymatic activity. We further show that QDs and fluorescent proteins can be conjugated together intracellularly with strong potential for live-cell imaging and biosensing applications.

Nanoparticle-based delivery of nitric oxide for therapeutic applications.

Nitric oxide (NO), a low molecular weight signaling molecule, plays critical roles in both cellular health and disease. There is continued interest in new modalities for the controlled therapeutic delivery of NO to cells and tissues. The physicochemical properties of NO (including its short half-life and on-demand synthesis at the point of function), however, pose considerable challenges for its specific and efficient delivery. Recently, a number of nanoparticle (NP)-based systems are described that address some of these issues by taking advantage of the unique attributes of the NP carrier to effect efficient NO delivery. This review highlights the progress that has been made over the past 5 years in the use of various constructs for the therapeutic delivery of NO.

This review details progress made over the past 5 years in the implementation of various nanoparticle (NP) bioconjugates for the therapeutic delivery of nitric oxide. Various NP formulations including liposomes, polymeric NPs, and hard NPs such as AuNPs and upconversion NPs are covered and we discuss the inherent advantages and challenges in using these materials for the controlled delivery of nitric oxide to cells and tissues.

Hybrid Nucleic Acid-Quantum Dot Assemblies as Multiplexed Reporter Platforms for Cell-Free Transcription Translation-Based Biosensors.

Cell-free synthetic biology has emerged as a valuable tool for the development of rapid, portable biosensors that can be readily transported in the freeze-dried form to the point of need eliminating cold chain requirements. One of the challenges associated with cell-free sensors is the ability to simultaneously detect multiple analytes within a single reaction due to the availability of a limited set of fluorescent and colorimetric reporters. To potentially provide multiplexing capabilities to cell-free biosensors, we designed a modular semiconductor quantum dot (QD)-based reporter platform that is plugged in downstream of the transcription-translation functionality in the cell-free reaction and which converts enzymatic activity in the reaction into distinct optical signals. We demonstrate proof of concept by converting restriction enzyme activity, utilized as our prototypical sensing output, into optical changes across several distinct spectral output channels that all use a common excitation wavelength. These hybrid Förster resonance energy transfer (FRET)-based QD peptide PNA-DNA-Dye reporters (QD-PDDs) are completely self-assembled and consist of differentially emissive QD donors paired to a dye-acceptor displayed on a unique DNA encoding a given enzyme's cleavage site. Three QD-based PDDs, independently activated by the enzymes BamHI, EcoRI, and NcoI, were prototyped in mixed enzyme assays where all three demonstrated the ability to convert enzymatic activity into fluorescent output. Simultaneous monitoring of each of the three paired QD-donor dye-acceptor spectral channels in cell-free biosensing reactions supplemented with added linear genes encoding each enzyme confirmed robust multiplexing capabilities for at least two enzymes when co-expressed. The modular QD-PDDs are easily adapted to respond to other restriction enzymes or even proteases if desired.