Search

Enzyme activity can be many times enhanced in configurations where they are displayed on a nanoparticle (NP) and this same format sometimes even provides access to channeling phenomena within multienzyme...

Interest in multi-enzyme synthesis outside of cells (in vitro) is becoming far more prevalent as the field of cell-free synthetic biology grows exponentially. Such synthesis would allow for complex chemical transformations based on the exquisite specificity of enzymes in a "greener" manner as compared to organic chemical transformations. Here, we describe how nanoparticles, and in this specific case-semiconductor quantum dots, can be used to both stabilize enzymes and further allow them to self-assemble into nanocomplexes that facilitate high-efficiency channeling phenomena. Pertinent protocol information is provided on enzyme expression, choice of nanoparticulate material, confirmation of enzyme attachment to nanoparticles, assay format and tracking, data analysis, and optimization of assay formats to draw the best analytical information from the underlying processes.

The SARS‐CoV‐2 virus binds to host cell surface ACE2 on the plasma membrane via the spike protein's receptor binding domain. Our work has resulted in the generation of a versatile imaging probe using recombinant Spike receptor binding domain conjugated to fluorescent quantum dots (QDs). This probe is capable of engaging in energy transfer quenching with ACE2‐conjugated gold nanoparticles enabling biochemical monitoring of binding. Neutralizing antibodies and recombinant human ACE2 blocked quenching, demonstrating a specific binding interaction. In cell‐based assays, we observed immediate binding of the probe on the cell surface of ACE2‐expressing cells followed by endocytosis. Neutralizing antibodies and ACE2‐Fc fully prevented binding and endocytosis with low nanomolar potency. Importantly, we can use this QD nanoparticle probe to identify and validate inhibitors of the SARS‐CoV‐2 Spike and ACE2 receptor binding in human cells. This work enables facile, rapid, and high‐throughput biochemical‐ and cell‐based screening of inhibitors for coronavirus Spike‐mediated cell recognition and entry.

The first step of SARS-CoV-2 infection is binding of the spike protein's receptor binding domain to the host cell's ACE2 receptor on the plasma membrane. Here, we have generated a versatile imaging probe using recombinant Spike receptor binding domain conjugated to fluorescent quantum dots (QDs). This probe is capable of engaging in energy transfer quenching with ACE2-conjugated gold nanoparticles to enable monitoring of the binding event in solution. Neutralizing antibodies and recombinant human ACE2 blocked quenching, demonstrating a specific binding interaction. In cells transfected with ACE2-GFP, we observed immediate binding of the probe on the cell surface followed by endocytosis. Neutralizing antibodies and ACE2-Fc fully prevented binding and endocytosis with low nanomolar potency. Importantly, we will be able to use this QD nanoparticle probe to identify and validate inhibitors of the SARS-CoV-2 Spike and ACE2 receptor binding in human cells. This work enables facile, rapid, and high-throughput cell-based screening of inhibitors for coronavirus Spike-mediated cell recognition and entry.

We demonstrate the viability of using ultra-thin sheets of microbially grown nanocellulose to build functional medical sensors. Microbially grown nanocellulose is an interesting alternative to plastics, as it is hydrophilic, biocompatible, porous, and hydrogen bonding, thereby allowing the potential development of new application routes. Exploiting the distinguishing properties of this material enables us to develop solution-based processes to create nanocellulose printed circuit boards, allowing a variety of electronics to be mounted onto our nanocellulose. As proofs of concept, we have demonstrated applications in medical sensing such as heart rate monitoring and temperature sensing&mdash, potential applications fitting the wide-ranging paradigm of a future where the Internet of Things is dominant.

Lipases are an important class of lipid hydrolyzing enzymes that play significant roles in many aspects of cell biology and digestion; they also have large roles in commercial food and biofuel preparation and are being targeted for pharmaceutical development. Given these, and many other biotechnological roles, sensitive and specific biosensors capable of monitoring lipase activity in a quantitative manner are critical. Here, we describe a Forster resonance energy transfer (FRET)-based biosensor that originates from a custom-synthesized ester substrate displaying a peptide at one end and a dye acceptor at the other. These substrates were ratiometrically self-assembled to luminescent semiconductor quantum dot (QD) donors by metal affinity coordination using the appended peptide's terminal hexahistidine motif to give rise to the full biosensing construct. This resulted in a high rate of FRET between the QD donor and the proximal substrate's dye acceptor. The lipase hydrolyzed the intervening target ester bond in the peptide substrate which, in turn, displaced the dye acceptor containing component and altered the rate of FRET in a concentration-dependent manner. Specifics of the substrate's stepwise synthesis are described along with the sensors assembly, characterization, and application in a quantitative proof-of-concept demonstration assay that is based on an integrated Michaelis-Menten kinetic approach. The utility of this unique nanoparticle-based architecture within a sensor configuration is then discussed.

Although a fundamental understanding of the pathogenicity of most biothreat agents has been elucidated and available treatments have increased substantially over the past decades, they still represent a significant public health threat in this age of (bio)terrorism, indiscriminate warfare, pollution, climate change, unchecked population growth, and globalization. The key step to almost all prevention, protection, prophylaxis, post-exposure treatment, and mitigation of any bioagent is early detection. Here, we review available methods for detecting bioagents including pathogenic bacteria and viruses along with their toxins. An introduction placing this subject in the historical context of previous naturally occurring outbreaks and efforts to weaponize selected agents is first provided along with definitions and relevant considerations. An overview of the detection technologies that find use in this endeavor along with how they provide data or transduce signal within a sensing configuration follows. Current "gold" standards for biothreat detection/diagnostics along with a listing of relevant FDA approved in vitro diagnostic devices is then discussed to provide an overview of the current state of the art. Given the 2014 outbreak of Ebola virus in Western Africa and the recent 2016 spread of Zika virus in the Americas, discussion of what constitutes a public health emergency and how new in vitro diagnostic devices are authorized for emergency use in the U.S. are also included. The majority of the Review is then subdivided around the sensing of bacterial, viral, and toxin biothreats with each including an overview of the major agents in that class, a detailed cross-section of different sensing methods in development based on assay format or analytical technique, and some discussion of related microfluidic lab-on-a-chip/point-of-care devices. Finally, an outlook is given on how this field will develop from the perspective of the biosensing technology itself and the new emerging threats they may face.

Cell-free synthetic biology relies on optimally exploiting enzymatic activity, and recent demonstrations that nanoparticle (NP) and DNA scaffolding can enhance enzyme activity suggest new avenues toward this. A modular architecture consisting of a DNA cage displaying semiconductor quantum dots (QDs) that, in turn, ratiometrically display the organophosphate hydrolase phosphotriesterase (PTE) was utilized as a model system. Increasing DNA cage concentration relative to QD-PTE and creating a dense composite enhanced PTE rates up to 12.5-fold, suggesting strong synergy between the NP and DNA components; this putatively arises from increased enzymatic stability and alleviation of its rate-limiting step. Such bioinorganic composites may offer new scaffolding approaches for synthetic biology.

Curved bilayer plates with soft and stiff segments are widely observed in nature, such as plant cell walls, insect exoskeletons and reptile skins. We report an unusual biaxial and bidirectional bending of microfabricated bilayer plates composed of a swellable, photopolymerized poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAM-AAc) layer and a regular array of SU-8, a stiff, non-swellable epoxy. Hydrogels such as pNIPAM-AAc exhibit large and reversible swelling in water in response to a temperature change through the lower critical solution temperature (LCST). The stimuli responsive behavior was harnessed in the composite structure containing materials with mismatched swelling and elastic properties to produce actuation and mechanical motion. The structure undergoes reversible bending along two different axes in response to a temperature cycle through the LCST. Cooling the patterned bilayer structure leads to bending upwards about one axis, while heating leads to bending downwards about a different axis, 9 0 o from the first. To understand the mechanism of this biaxial bending behavior, we developed a finite element model of the patterned bilayer structure. A constitutive model that combined the hyperelastic and swelling behavior was used to describe the thermoresponsive hydrogel. The model was applied to investigate the effects of geometric factors of the patterned bilayer on the bending behavior of the composite structure.

Combining biomolecules such as enzymes with nanoparticles has much to offer for creating next generation synergistically functional bionanomaterials. However, almost nothing is known about how these two disparate components interact at this critical biomolecular-materials interface to give rise to improved activity and emergent properties. Here we examine how the nanoparticle surface can influence and increase localized enzyme activity using a designer experimental system consisting of trypsin proteolysis acting on peptide-substrates displayed around semiconductor quantum dots (QDs). To minimize the complexity of analyzing this system, only the chemical nature of the QD surface functionalizing ligands were modified. This was accomplished by synthesizing a series of QD ligands that were either positively or negatively charged, zwitterionic, neutral, and with differing lengths. The QDs were then assembled with different ratios of dye-labeled peptide substrates and exposed to trypsin giving rise to progress curves that were monitored by Förster resonance energy transfer (FRET). The resulting trypsin activity profiles were analyzed in the context of detailed molecular dynamics simulations of key interactions occurring at this interface. Overall, we find that a combination of factors can give rise to a localized activity that was 35-fold higher than comparable freely diffusing enzyme-substrate interactions. Contributing factors include the peptide substrate being prominently displayed extending from the QD surface and not sterically hindered by the longer surface ligands in conjunction with the presence of electrostatic and other productive attractive forces between the enzyme and the QD surface. An intimate understanding of such critical interactions at this interface can produce a set of guidelines that will allow the rational design of next generation high-activity bionanocomposites and theranostics.

The rapid detection of organophosphates (OPs), a class of strong neurotoxins, is critically important for monitoring acute insecticide exposure and potential chemical warfare agent use. Herein, we improve the enzymatic activity of a phosphotriesterase trimer (PTE3), an enzyme that selectively recognizes OPs directly, by conjugation with distinctly sized (i.e., 5, 10, and 20 nm diameter) gold nanoparticles (AuNPs). The number of enzymes immobilized on the AuNP was controlled by conjugating increasing molar ratios of PTE3 onto the AuNP surface via metal affinity coordination. This occurs between the PTE3-His6 termini and the AuNP-displayed Ni2+-nitrilotriacetic acid end groups and was confirmed with gel electrophoresis. The enzymatic efficiency of the resultant PTE3–AuNP bioconjugates was analyzed via enzyme progress curves acquired from two distinct assay formats that compared free unbound PTE3 with the following PTE3–AuNP bioconjugates: (1) fixed concentration of AuNPs while increasing the bioconjugate molar ratio of PTE3 displayed around the AuNP and (2) fixed concentration of PTE3 while increasing the bioconjugate molar ratio of PTE3–AuNP by decreasing the AuNP concentration. Both assay formats monitored the absorbance of p-nitrophenol that was produced as PTE3 hydrolyzed the substrate paraoxon, a commercial insecticide and OP nerve agent simulant. Results demonstrate a general equivalent trend between the two formats. For all experiments, a maximum enzymatic velocity (Vmax) increased by 17-fold over free enzyme for the lowest PTE3–AuNP ratio and the largest AuNP (i.e., ratio of 1 : 1, 20 nm dia. AuNP). This work provides a route to improve enzymatic OP detection strategies with enzyme–NP bioconjugates.

Significant efforts are being undertaken to optimize the cargo carrying capacity and especially the cellular delivery efficiency of functionalized nanoparticles for applications in biological research and pharmacological delivery. One approach to increasing nanoparticle surface cargo display capacity is to decrease the number of moieties required for mediating cellular delivery by improving their efficiency. We describe a series of multivalent cell penetrating peptide (CPP) dendrimers that facilitate rapid cellular delivery of prototypical nanoparticle-semiconductor quantum dots (QDs). The modular CPP dendrimers were assembled through an innovative convergent oxime ligation strategy between (Arg9)n motifs and a dendritic QD-coordination scaffold. Dendrimeric peptides sequentially incorporate a terminal (His)6 motif for metal-affinity QD coordination, a Pro9 spacer, a branching poly-lysine scaffold, and wedged display of (Arg9)n binding motifs with n = 1×, 2×, 4×, 8×, 16× multivalency. QD dendrimer display capacity was estimated using structural simulations and QD–(Arg9)1–16 conjugates characterized by dynamic light scattering along with surface plasmon resonance-based binding assays to heparan sulfate proteoglycan surfaces. Cellular uptake via endocytosis was confirmed and peptide delivery kinetics investigated as a function of QD–(Arg9)1–16 conjugate exposure time and QD assembly ratio where cellular viability assays reflected no overt cytotoxicity. The ability of single dendrimer conjugates to facilitate cellular uptake was confirmed for QD–(Arg9)2–16 repeats along with the ability to deliver >850 kDa of protein cargo per QD. Minimizing the number of CPPs required for cellular uptake is critical for expanding nanoparticle cargo carrying capacity and can allow for inclusion of additional sensors, therapeutics and contrast agents on their surface.

Accumulating studies by many groups have found consistent enhancement in a wide variety of enzyme activities when they are displayed around nanoparticles. However, the underlying mechanism(s) that give rise to this phenomenon are still largely unknown. Herein, we develop a detailed reaction scheme that considers many of the various possible interactions between a substrate and a given enzyme-nanoparticle bioconjugate. The properties and some functional predictions that emanate from the reaction scheme were then tested using a model system where the homotetrameric beta-galactosidase enzyme complex was assembled with luminescent semiconductor nanocrystalline quantum dots displayed around its periphery. This type of assembly occurs as the ∼465 kDa enzyme complex is significantly larger than the 4.2 nm diameter green emitting quantum dots utilized. This unique architecture, in conjunction with the fact that this enzyme functions at or near the diffusion limit, provided a unique opportunity to selectively probe certain aspects of enzyme enhancement when attached to a nanoparticle with minimal potential perturbations to the native enzyme structure. Experimental assays were conducted where both free enzymes and quantum dot-decorated enzymes were compared directly in side-by-side samples and included formats where the kinetic processes were challenged with increasing viscosity and competitive inhibitors. The results strongly suggest that it is possible for there to be significant enhancements in an enzyme's catalytic rate or kcat after attachment to a nanoparticle even when it is apparently diffusion limited without requiring any gross changes to the enzyme's structure. A discussion of how this reaction scheme and model can be applied to other systems is provided.

Enhancements in enzymatic catalytic activity are frequently observed when an enzyme is displayed on a nanoparticle (NP) surface. The exact mechanisms of how this unique interfacial environment gives rise to this phenomenon are still not understood, although evidence suggests that it can help alleviate some of the enzyme's rate-limiting mechanistic steps. The physicochemical limitations that govern when this process arises are also not known including, in particular, the range of NP size and curvature that are associated with it. To investigate the latter, we undertook a case study using the enzyme phosphotriesterase (PTE) and a series of differentially sized gold NPs (AuNPs). PTE, expressed with a terminal hexahistidine sequence, was ratiometrically coordinated to a series of increasing size AuNPs (diameter ≃ 1.5, 5, 10, 20, 55, 100 nm) surface-functionalized with Ni

We present the first demonstration of a fully-flexible, self-powered glucose indicator system that synergizes two flexible electronic technologies: a flexible self-powering unit in the form of a biofuel cell, with a flexible electronic device - a circuit-board decal fabricated with biocompatible microbial nanocellulose. Our proof-of-concept device, comprising an enzymatic glucose fuel cell, glucose sensor and a LED indicator, does not require additional electronic equipment for detection or verification; and the entire structure collapses into a microns-thin, self-adhering, single-centimeter-square decal, weighing less than 40 mg. The flexible glucose indicator system continuously operates a light emitting diode (LED) through a capacitive charge/discharge cycle, which is directly correlated to the glucose concentration. Our indicator was shown to operate at high sensitivity within a linear glucose concentration range of 1 mM–45 mM glucose continuously, achieving a 1.8 VDC output from a flexible indicator system that deliver sufficient power to drive an LED circuit. Importantly, the results presented provide a basis upon which further development of indicator systems with biocompatible diffusing polymers to act as buffering diffusion barriers, thereby allowing them to be potentially useful for low-cost, direct-line-of-sight applications in medicine, husbandry, agriculture, and the food and beverage industries.

A web-based resource for meta-analysis of nanomaterials toxicity is developed whereby the utility of Bayesian networks (BNs) is illustrated for exploring the cellular toxicity of Cd-containing quantum dots (QDs). BN models are developed based on a dataset compiled from 517 publications comprising 3028 cell viability data samples and 837 IC50 values. BN QD toxicity (BN-QDTox) models are developed using both continuous (i.e., numerical) and categorical attributes. Using these models, the most relevant attributes identified for correlating IC50 are: QD diameter, exposure time, surface ligand, shell, assay type, surface modification, and surface charge, with the addition of QD concentration for the cell viability analysis. Data exploration via BN models further enables identification of possible association rules for QDs cellular toxicity. The BN models as web-based applications can be used for rapid intelligent query of the available body of evidence for a given nanomaterial and can be readily updated as the body of knowledge expands.

Bio-inspired, hybrid architectures employing quantum dots (QDs) appended with functionally active biomolecules such as enzymes have the potential to be utilized in numerous applications. Some examples include nanosensors for medical diagnostics, chemical/biological threat detection, as well as “bio-factories” in complex industrial synthetic processes. The main advantage in creating these nanofactories is increased rates in catalysis and efficiency when enzymes are associated with nanoscaffolds, as shown in numerous studies. However, the mechanism for this enhancement remains elusive. Gaining a fundamental, mechanistic understanding of enzyme-QD nanostructures is important in the development of numerous device applications. In this work, we review an array of enzymes attached to QDs and generate a hypothesis in regards to the unique architecture of the enzyme-nanoparticle (NP) construct that leads to increases in catalysis. We highlight work with phosphotiresterase (PTE) attached to two distinctly sized QDs in neutralizing a simulant nerve agent, as well as in other enzyme systems.

We discuss the self-folding of patterned metallic sheets using both differential stress and surface forces. The advantageous characteristics of the technique include, (a) The creation of 3D patterned corrugated metamaterials with pattern resolution limited only by that of planar lithography. Since planar lithography is highly versatile, a variety of patterns with different sizes and shapes can be formed. (b) The hands-free and wire-free self-folding of these materials use two orthogonal forces derived from the release of residual stress and the minimization of surface tension. Hence, this process is highly parallel and scalable allowing such materials to be mass produced. (c) Finally, the edges of the materials self-align and seal due to capillary forces of the liquid hinges—this self-sealing enhances overall rigidity and strength of the materials. Consequently, the self-folding of patterned and sealed “egg-crate” shaped metamaterials was realized. Patterns were incorporated in the form of “smart” patches on the walls of the egg-crates which can be selectively functionalized with biomolecules. Apart from the intellectual appeal of these hands-free, self-sealing materials, we envision applicability of these egg-crate like microstructures in lab-on-a-chip assays as functionalized microwells and as light weight mechanical metamaterials.

The Bacillus collagen-like protein of anthracis (BclA), found in Bacillus anthracis spores, is an attractive target for immunoassays. Previously, using phage display we had selected llama-derived single-domain antibodies that bound to B. anthracis spore proteins including BclA. Single-domain antibodies (sdAbs), the recombinantly expressed heavy domains from the unique heavy-chain-only antibodies found in camelids, provide stable and well-expressed binding elements with excellent affinity. In addition, sdAbs offer the important advantage that they can be tailored for specific applications through protein engineering. A fusion of a BclA targeting sdAb with the enzyme Beta galactosidase (&beta, gal) would enable highly sensitive immunoassays with no need for a secondary reagent. First, we evaluated five anti-BclA sdAbs, including four that had been previously identified but not characterized. Each was tested to determine its binding affinity, melting temperature, producibility, and ability to function as both capture and reporter in sandwich assays for BclA. The sdAb with the best combination of properties was constructed as a fusion with &beta, gal and shown to enable sensitive detection. This fusion has the potential to be incorporated into highly sensitive assays for the detection of anthrax spores.

Multistep enzymatic cascades are becoming more prevalent in industrial settings as engineers strive to synthesize complex products and pharmaceuticals in economical, environmentally friendly ways. Previous work has shown that immobilizing enzymes on nanoparticles can enhance their activity significantly due to localized interfacial effects, and this enhancement remains in place even when that enzyme's activity is coupled to another enzyme that is still freely diffusing. Here, we investigate the effects of displaying two enzymes with coupled catalytic activity directly on the same nanoparticle surface. For this, the well-characterized enzymes pyruvate kinase (PykA) and lactate dehydrogenase (LDH) were utilized as a model system; they jointly convert phosphoenolpyruvate to lactate in two sequential steps as part of downstream glycolysis. The enzymes were expressed with terminal polyhistidine tags to facilitate their conjugation to semiconductor quantum dots (QDs) which were used here as prototypical nanoparticles. Characterization of enzyme coassembly to two different sized QDs showed a propensity to cross-link into nanoclusters consisting of primarily dimers and some trimers. Individual and joint enzyme activity in this format was extensively investigated in direct comparison to control samples lacking the QD scaffolds. We found that QD association enhances LDH activity by50-fold and its total turnover by at least 41-fold, and that this high activation appears to be largely due to stabilization of its quarternary structure. When both enzymes are simultaneously bound to the QD surfaces, their colocalization leads to100-fold improvements in the overall rates of coupled activity. Experimental results in conjunction with detailed kinetic simulations provide evidence that this significant improvement in coupled activity is partially attributable to a combination of enhanced enzymatic activity and stabilization of LDH. More importantly, experiments aimed at disrupting channeled processes and further kinetic modeling suggest that the bulk of the performance enhancement arises from intermediary "channeling" between the QD-colocalized enzymes. A full understanding of the underlying processes that give rise to such enhancements from coupled enzymatic activity on nanoparticle scaffolds can provide design criteria for improved biocatalytic applications.

Enhancement in enzymatic activity after attachment to nanoparticle surfaces has been observed in numerous enzyme systems, although the underlying mechanism for these enhancements remains largely unknown. This work explores the utility of a model based on a reaction scheme that takes into account some of the many interactions between substrate, product, and nanoparticle that can occur. This model was utilized to make predictions about the type of behavior that should manifest itself with quantum dots peripherally displayed around beta-galactosidase (&beta-gal) and confirmed empirically. &beta-gal is a homotetrameric enzyme which at ~465 kDa is significantly larger than the 4.2 nm diameter green emitting quantum dots utilized to decorate its periphery. Because &beta-gal operates near the diffusion limit, this provides an opportunity to selectively investigate certain aspects of enzyme enhancement when attached to a nanoparticle with minimal perturbation to the native enzyme structure. Enzymatic assays were performed with both free enzyme and quantum dot-decorated enzymes in a side-by-side format where kinetic processes were challenged by increasing viscosity with glycerol and competitive inhibitors such as lactose. The results from this model suggest it is possible to achieve significant enhancements in a diffusion limited enzyme’s catalytic rate ( k cat ) after NP attachment without substantial changes to the enzyme’s structure or function. Because cell free synthetic biology is gaining importance, this approach will yield insights on how enzymes can be utilized ex vivo and how being attached to NP scaffolds yields kinetic enhancement, possibly through enhanced product dissociation.

Solution phase printing of graphene-based electrodes has recently become an attractive low-cost, scalable manufacturing technique to create in-field electrochemical biosensors. Here, we report a graphene-based electrode developed via inkjet maskless lithography (IML) for the direct and rapid monitoring of triple-O linked phosphonate organophosphates (OPs); these constitute the active compounds found in chemical warfare agents and pesticides that exhibit acute toxicity as well as long-term pollution to soils and waterways. The IML-printed graphene electrode is nano/microstructured with a 1000 mW benchtop laser engraver and electrochemically deposited platinum nanoparticles (dia. ∼25 nm) to improve its electrical conductivity (sheet resistance decreased from ∼10 000 to 100 Ω/sq), surface area, and electroactive nature for subsequent enzyme functionalization and biosensing. The enzyme phosphotriesterase (PTE) was conjugated to the electrode surface via glutaraldehyde cross-linking. The resulting biosensor was able to rapidly measure (5 s response time) the insecticide paraoxon (a model OP) with a low detection limit (3 nM), and high sensitivity (370 nA/μM) with negligible interference from similar nerve agents. Moreover, the biosensor exhibited high reusability (average of 0.3% decrease in sensitivity per sensing event), stability (90% anodic current signal retention over 1000 s), longevity (70% retained sensitivity after 8 weeks), and the ability to selectively sense OP in actual soil and water samples. Hence, this work presents a scalable printed graphene manufacturing technique that can be used to create OP biosensors that are suitable for in-field applications as well as, more generally, for low-cost biosensor test strips that could be incorporated into wearable or disposable sensing paradigms.

As a specific example of the enhancement of enzymatic activity that can be induced by nanoparticles, we investigate the hydrolysis of the organophosphate paraoxon by phosphotriesterase (PTE) when the latter is displayed on semiconductor quantum dots (QDs). PTE conjugation to QDs underwent extensive characterization including structural simulations, electrophoretic mobility shift assays, and dynamic light scattering to confirm orientational and ratiometric control over enzyme display which appears to be necessary for enhancement. PTE hydrolytic activity was then examined when attached to ca. 4 and 9 nm diameter QDs in comparison to controls of freely diffusing enzyme alone. The results confirm that the activity of the QD conjugates significantly exceeded that of freely diffusing PTE in both initial rate (∼4-fold) and enzymatic efficiency (∼2-fold). To probe kinetic acceleration, various modified assays including those with increased temperature, presence of a competitive inhibitor, and increased viscosity were undertaken to measure the activation energy and dissociation rates. Cumulatively, the data indicate that the higher activity is due to an acceleration in enzyme-product dissociation that is presumably driven by the markedly different microenvironment of the PTE-QD bioconjugate's hydration layer. This report highlights how a specific change in an enzymatic mechanism can be both identified and directly linked to its enhanced activity when displayed on a nanoparticle. Moreover, the generality of the mechanism suggests that it could well be responsible for other examples of nanoparticle-enhanced catalysis.

Hydrogels such as poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAM-AAc) can be photopatterned to create a wide range of actuatable and self-folding microstructures. Mechanical motion is derived from the large and reversible swelling response of this cross-linked hydrogel in varying thermal or pH environments. This action is facilitated by their network structure and capacity for large strain. However, due to the low modulus of such hydrogels, they have limited gripping ability of relevance to surgical excision or robotic tasks such as pick-and-place. Using experiments and modeling, we design, fabricate, and characterize photopatterned, self-folding functional microgrippers that combine a swellable, photo-cross-linked pNIPAM-AAc soft-hydrogel with a nonswellable and stiff segmented polymer (polypropylene fumarate, PPF). We also show that we can embed iron oxide (Fe2O3) nanoparticles into the porous hydrogel layer, allowing the microgrippers to be responsive and remotely guided using magnetic fields. Using finite element models, we investigate the influence of the thickness and the modulus of both the hydrogel and stiff polymer layers on the self-folding characteristics of the microgrippers. Finally, we illustrate operation and functionality of these polymeric microgrippers for soft robotic and surgical applications.

Enzymes provide the critical means by which to catalyze almost all biological reactions in a controlled manner. Methods to harness and exploit their properties are of strong current interest to the growing field of biotechnology. In contrast to depending upon recombinant genetic approaches, a growing body of evidence suggests that apparent enzymatic activity can be enhanced when located at a nanoparticle interface. We use semiconductor quantum dots (QDs) as a well-defined and easily bioconjugated nanoparticle along with Escherichia coli-derived alkaline phosphatase (AP) as a prototypical enzyme to seek evidence for this process in a de novo model system. We began by first assessing whether the relatively large dimeric AP protein (∼100 kDa) can be assembled onto two differentially sized green and red CdSe/ZnS core/shell QDs in a ratiometric and structurally controlled manner; such assembly is necessary to minimize heterogeneity within the bioconjugate and provide intimate control over the experimental form...

Ionically crosslinked alginate hydrogels have been extensively explored for encapsulation and immunoisolation of living cells/tissues to develop implantable cell therapies, such as islet encapsulation for bioartificial pancreas. Chemical instability of these hydrogels during long-term implantation hinders the development of viable cell therapy. The exchange between divalent crosslinking ions (e.g., Ca(+2) ) with monovalent ions from physiological environment causes alginate hydrogels to degrade, resulting in exposure of the donor tissue to the host's immune system and graft failure. The goal of this study was to improve stability of alginate hydrogels by utilizing covalent "click" crosslinking while preserving other biomedically viable hydrogel properties. Alginate was first functionalized to contain either pendant alkyne or azide functionalities, and subsequently reacted via "click" chemistry to form "click" gel capsules. Alginate functionalization was confirmed by NMR and gel permeation chromatography. When compared with Ca(+2) capsules, "click" capsules exhibited superior stability in ionic media, while showing higher permeability to small size diffusants and similar molecular weight cut-off and water swelling. Physicochemical properties of "click" alginate hydrogels demonstrate their potential utility for therapeutic cell encapsulation and other biomedical applications.

People can get oral cancers from UV (290-400 nm) exposures. Besides high outdoor UV exposures, high indoor UV exposures to oral tissues can occur when consumers use UV-emitting tanning devices to either tan or whiten their teeth. We compared the carcinogenic risks of skin to oral tissue cells after UVB (290-320 nm) exposures using commercially available 3D-engineered models for human skin (EpiDerm™), gingival (EpiGing™) and oral (EpiOral™) tissues. To compare the relative carcinogenic risks, we investigated the release of cytokines, initial DNA damage in the form of cyclobutane pyrimidine dimers (CPDs), repair of CPDs and apoptotic cell numbers. We measured cytokine release using cytometric beads with flow cytometry and previously developed a fluorescent immunohistochemical assay to quantify simultaneously CPD repair rates and apoptotic cell numbers. We found that interleukin-8 (IL-8) release and the initial CPDs are significantly higher, whereas the CPD repair rates and apoptotic cell numbers are significantly lower for oral compared with skin tissue cells. Thus, the increased release of the inflammatory cytokine IL-8 along with inefficient CPD repair and decreased death rates for oral compared with skin tissue cells suggests that mutations are accumulating in the surviving population of oral cells increasing people's risks for getting oral cancers.

With continued advancements in nanoparticle (NP) synthesis and in the interfacing of NPs with biological systems has come the exponential growth in the use of NPs for therapeutic drug delivery and imaging applications. In recent years, the advent of NP multifunctionality-the ability to perform multiple, disparate functions on a single NP platform-has garnered much excitement for the potential realization of highly functional NP-mediated drug delivery for use in the clinical setting. This Overview will survey the current state of the art (reports published within the last 5 years) of multifunctional NPs for therapeutic drug delivery, imaging or a combination thereof. We provide extensive examples of both soft (micelles, liposomes, polymeric NPs) and hard (noble metals, quantum dots, metal oxides) NP formulations that have been used for multimodal drug delivery and imaging. The criteria for inclusion, herein, is that there must be at least two therapeutic drug cargos or imaging agents or a combination of the two. We next offer an assessment of the cytotoxicity of therapeutic NP constructs in biological systems. We then conclude with a forward-looking perspective on how we expect this field to develop in the coming years. WIREs Nanomed Nanobiotechnol 2017, 9:e1466. doi: 10.1002/wnan.1466 For further resources related to this article, please visit the WIREs website.

A primary envisioned use for nanoparticles (NPs) in a cellular context is for controlled drug delivery where the full benefit of NP attributes (small size, large drug cargo loading capacity) can improve the pharmacokinetics of the drug cargo. This requires the ability to controllably manipulate the release of the drug cargo from the NP vehicle or ‘controlled actuation’. In this review, we highlight new developments in this field from 2013 to 2015. The number and breadth of reports are a testament to the significant advancements made in this field over this time period. We conclude with a perspective of how we envision this field to continue to develop in the years to come.

Semiconductor quantum dots (QDs) serve as a valuable platform for understating the intricacies of nanoparticle cellular uptake and fate for the development of theranostics. Developing novel internalization peptides that maximize cellular uptake while minimizing the amount of peptide is important to allow space on the nanoparticle for other cargo (e.g. drugs). We have designed a range of branched, dendritic internalization peptides composed of polyarginine (Arg 9 ) branches (1 to 16 repeats) attached a dendritic wedge based on the sequence WP9G2H6. By attaching these branched dendritic peptides to QD’s, we can study the influence of branching on cellular uptake as a function of time, ratio, and degree of branching.

Rational design of enzyme-nanoparticle hybrids is still in its infancy and the design is often inspired by potential access to many beneficial sensing properties such as increased stability, sensitivity, and even enhanced enzyme activities in specific cases. Deriving quantitative kinetic data from these constructs is not trivial, however, since the intrinsic design gives rise to unique properties that can influence the enzymatic assays that are central to the application of the hybrids. Here, we present two distinct assay methodologies for following the kinetic activity of composite enzyme-nanoparticle constructs. We utilize luminescent semiconductor nanocrystals or quantum dots (QDs) as the prototypical nanoparticulate platform for these sensing formats and target proteolytic enzyme activity as the main assay. The first assay is analogous to most current enzymatic assays and is designed to compare QD-enzyme constructs; this format is based on utilizing a fixed concentration of enzyme displayed on the QD and excess substrate in the solution, and the analysis utilizes data from initial velocities. The second assay is designed to analyze kinetics using a QD-substrate construct, in which the enzyme and QD interactions are short lived. Here, the nanoparticle-substrate concentration is held constant and exposed to increasing concentrations of the enzyme in solution. This later methodology is based on a fluorescent ratiometric signal that follows the entire progress curve of the enzyme reaction. A comparison of these two different assays of the series of enzyme-nanoparticle and substrate-nanoparticle constructs provides deeper insight into the enzyme kinetics of these hybrids, while still testing of individual variables within a given format, to allow for further optimization within each set.

Enzymes are important players in multiple applications, be it bioremediation, biosynthesis, or as reporters. The business of catalysis and inhibition of enzymes is a multibillion dollar industry and understanding the kinetics of commercial enzymes can have a large impact on how these systems are optimized. Recent advances in nanotechnology have opened up the field of nanoparticle (NP) and enzyme conjugates and two principal architectures for NP conjugate systems have been developed. In the first example the enzyme is bound to the NP in a persistent manner, here we find that key factors such as directed enzyme conjugation allow for enhanced kinetics. Through controlled comparative experiments we begin to tease out specific mechanisms that may account for the enhancement. The second system is based on dynamic interactions of the enzymes with the NP. The enzyme substrate is bound to the NP and the enzyme is free in solution. Here again we find that there are many variables , such as substrate positioning and NP selection, that modify the kinetics.

Nanosensors employing quantum dots (QDs) with appended biofunctional moieties offer tremendous promise for disease surveillance/diagnostics and chemical/biological threat activity. Their small size permits cell penetration and their inherent photochemical properties are well-suited for rapid, optical measurement. The effectiveness of enzymes immobilized on QDs, however, are not completely understood, hindering development of chemical/biological sensors and remediation materials. Here, we analyze enzyme effectiveness for the neutralization of a simulant nerve agent when attached to two distinctly-sized QDs. Two sizes of QDs, 525 or 625 nm, were appended with DHLA ligands to improve aqueous stability and prevent aggregation. Various molar ratios of de novo phosphotriesterase trimer (PTE3) were rapidly self-assembled via spontaneous metal coordination of the PTE oligohistidine tag onto the Zn2+-rich QD surface. PTE catalyzes the detoxification of organophosphate pesticides (e.g, paraoxon, an analog of sarin) to p-nitrophenol whose absorbance can be measured at 405 nm. The optimal ratio of PTE3 to 525 nm and 625 nm QD’s was determined to be 12 and 24, respectively. The enhanced enzyme performance in both cases is most likely due to increased enzyme-substrate interactions from improvements in enzyme orientation, enzyme density, and substrate diffusion on or near the QD. Development of these nansosensors as optical-based biosensors (e.g., within compact microfluidic devices) may greatly improve the sensitivity of conventional biological/chemical detection schemes.

Phosphotriesterase was engineered into a spontaneously forming trimer by appending it to a synthetic collagen-like triple-helix motif. Enzymatic hydrolysis of the insecticide and organophosphate nerve agent simulant paraoxon was then examined. Assembling the phosphotriesterase trimer onto semiconductor quantum dots increased the enzyme's catalytic rate and efficiency.

The controlled delivery of nanomaterials to the plasma membrane is critical for the development of nanoscale probes that can eventually enable cellular imaging and analysis of membrane processes. Chief among the requisite criteria are delivery/targeting modalities that result in the long-term residence (e.g., days) of the nanoparticles on the plasma membrane while simultaneously not interfering with regular cellular physiology and homeostasis. Our laboratory has developed a suite of peptidyl motifs that target semiconductor nanocrystals (quantum dots (QDs) to the plasma membrane where they remain resident for up to three days. Notably, only small a percentage of the QDs are endocytosed over this time course and cellular viability is maintained. This talk will highlight the utility of these peptide-QD constructs for cellular imaging and analysis.

Nanoparticle (NP) display potentially offers a new way to both stabilize and, in many cases, enhance enzyme activity over that seen for native protein in solution. However, the large, globular and sometimes multimeric nature of many enzymes limits their ability to attach directly to the surface of NPs, especially when the latter are colloidally stabilized with bulky PEGylated ligands. Engineering extended protein linkers into the enzymes to achieve direct attachment through the PEG surface often detrimentally alters the enzymes catalytic ability. Here, we demonstrate an alternate, hybrid biomaterials-based approach to achieving directed enzyme assembly on PEGylated NPs. We self-assemble a unique architecture consisting of a central semiconductor quantum dot (QD) scaffold displaying controlled ratios of extended peptide-DNA linkers which penetrate through the PEG surface to directly couple enzymes to the QD surface. As a test case, we utilize phosphotriesterase (PTE), an enzyme of bio-defense interest due to its ability to hydrolyze organophosphate nerve agents. Moreover, this unique approach still allows PTE to maintain enhanced activity while also suggesting the ability of DNA to enhance enzyme activity in and of itself.

Contamination and adulterants in both naturally derived and synthetic drugs pose a serious threat to the worldwide medical community. Developing rapid and sensitive sensors/devices to detect these hazards is thus a continuing need. We describe a hydrophilic semiconductor quantum dot (QD)-peptide Förster resonance energy transfer (FRET) nanosensor for monitoring the activity of kallikrein, a key proteolytic enzyme functioning at the initiation of the blood clotting cascade. Kallikrein is also activated by the presence of an oversulfated contaminant recently found in preparations of the drug heparin. Quantitatively monitoring the activity of this enzyme within a nanosensor format has proven challenging because of inherent steric and kinetic considerations. Our sensor is designed around a central QD donor platform which displays controlled ratios of a modular peptidyl substrate. This peptide, in turn, sequentially expresses a terminal oligohistidine motif that mediates the rapid self-assembly of peptides to the QD surface, a linker-spacer sequence to extend the peptide away from the QD surface, a kallikrein recognized-cleavage site, and terminates in an acceptor dye-labeling site. Hydrophilic QDs prepared with compact, zwitterionic surface coatings were first evaluated for their ability to self-assemble the dye-labeled peptide substrates. An optimized two-step protocol was then utilized where high concentrations of peptide were initially digested with purified human kallikrein and samples collected at distinct time points were subsequently diluted into QD-containing solutions for assaying. This sensor provided a quantitative FRET-based readout for monitoring kallikrein activity and comparison to a calibration curve allowed estimation of the relevant Michaelis-Menten kinetic descriptors. The results further suggest that almost any protease should be amenable to a QD-based FRET assay format with appropriate design considerations.

We report on a therapeutic approach using thermo-responsive multi-fingered drug eluting devices. These therapeutic grippers referred to as theragrippers are shaped using photolithographic patterning and are composed of rigid poly(propylene fumarate) segments and stimuli-responsive poly(N-isopropylacrylamide-co-acrylic acid) hinges. They close above 32 °C allowing them to spontaneously grip onto tissue when introduced from a cold state into the body. Due to porosity in the grippers, theragrippers could also be loaded with fluorescent dyes and commercial drugs such as mesalamine and doxorubicin, which eluted from the grippers for up to seven days with first order release kinetics. In an in vitro model, theragrippers enhanced delivery of doxorubicin as compared to a control patch. We also released theragrippers into a live pig and visualized release of dye in the stomach. The design of such tissue gripping drug delivery devices offers an effective strategy for sustained release of drugs with immediate applicability in the gastrointestinal tract.

Ionically crosslinked alginate hydrogels have been extensively explored for encapsulation and immunoisolation of living cells/tissues to develop implantable cell therapies, such as islet encapsulation for bioartificial pancreas. Chemical instability of these hydrogels during long-term implantation hinders the development of viable cell therapy. The exchange between divalent crosslinking ions (e.g., Ca(+2) ) with monovalent ions from physiological environment causes alginate hydrogels to degrade, resulting in exposure of the donor tissue to the host's immune system and graft failure. The goal of this study was to improve stability of alginate hydrogels by utilizing covalent "click" crosslinking while preserving other biomedically viable hydrogel properties. Alginate was first functionalized to contain either pendant alkyne or azide functionalities, and subsequently reacted via "click" chemistry to form "click" gel capsules. Alginate functionalization was confirmed by NMR and gel permeation chromatography. When compared with Ca(+2) capsules, "click" capsules exhibited superior stability in ionic media, while showing higher permeability to small size diffusants and similar molecular weight cut-off and water swelling. Physicochemical properties of "click" alginate hydrogels demonstrate their potential utility for therapeutic cell encapsulation and other biomedical applications.

The biological applications of luminescent semiconductor quantum dots (QDs) continue to grow at a nearly unabated pace. This growth is driven, in part, by their unique photophysical and physicochemical properties which have allowed them to be used in many different roles in cellular biology including: as superior fluorophores for a wide variety of cellular labeling applications; as active platforms for assembly of nanoscale sensors; and, more recently, as a powerful tool to understand the mechanisms of nanoparticle mediated drug delivery. Given that controlled cellular delivery is at the intersection of all these applications, the latest progress in delivering QDs to cells is examined here. A brief discussion of relevant considerations including the importance of materials preparation and bioconjugation along with the continuing issue of endosomal sequestration is initially provided for context. Methods for the cellular delivery of QDs are then highlighted including those based on passive exposure, facilitated strategies that utilize peptides or polymers and fully active modalities such as electroporation and other mechanically based methods. Following on this, the exciting advent of QD cellular delivery using multiple or combined mechanisms is then previewed. Several recent methods reporting endosomal escape of QD materials in cells are also examined in detail with a focus on the mechanisms by which access to the cytosol is achieved. The ongoing debate over QD cytotoxicity is also discussed along with a perspective on how this field will continue to evolve in the future.