Corked Microcapsules Enabling Controlled Ultrasound-Mediated Protein Delivery.

While ultrasound represents a facile, portable, and noninvasive trigger for drug delivery vehicles, most reported ultrasound-triggered drug delivery vehicles predominately present "burst" release profiles that are hard to control after the initial activation stimulus. Herein, we report a submerged electrospraying technique to fabricate protein-loaded microcapsules in which silica "corks" are embedded within the microcapsule shell. Upon the application of an ultrasound trigger, the corks can be perturbed within the shell, allowing for the release of the protein payload through a phantom tissue mimic to a degree proportional to the number/time of pulses applied. Specifically, multiple ultrasound pulses were shown to enable a 15- to 23-fold increase in the rate of release of the model bovine serum albumin protein payload relative to no ultrasound being applied, with release returning to a lower level when the ultrasound stimulus was removed. Coupled with the low cytotoxicity of the vehicle components, the corked microcapsules show promise for expanding the potential to use ultrasound to facilitate both on-demand and pulsatile release profiles.

Electrospun "Hard-Soft" Interpenetrating Nanofibrous Tissue Scaffolds Facilitating Enhanced Mechanical Strength and Cell Proliferation.

"Soft" hydrogel-based macroporous scaffolds have been widely used in tissue engineering and drug delivery applications due to their hydrated interfaces and macroporous structures, but have drawbacks related to their weak mechanics and often weak adhesion to cells. In contrast, "hard" poly(caprolactone) (PCL) electrospun fibrous networks have desirable mechanical strength and ductility but offer minimal interfacial hydration and thus limited capacity for cell proliferation. Herein, we demonstrate the fabrication of interpenetrating nanofibrous networks based on coelectrospun PCL and poly(oligoethylene glycol methacrylate) (POEGMA) nanofibers that exhibit the mechanical benefits of PCL but the interfacial hydration benefits of hydrogels. The electrospinning process results in partially aligned but interpenetrating fiber network with minimal internal phase separation, leading to anisotropic but strong mechanical properties even in the hydrated state; apparent ultimate tensile strengths of the swollen scaffolds ranged from 429 ± 39 kPa in the direction of fiber alignment (longitudinal) to 86 ± 25 kPa perpendicular to fiber alignment (cross-longitudinal), typical of PCL-based scaffolds and enabling efficient suture retention in different directions. However, contact angle measurements indicate hydrogel-like interfacial properties due to the presence of the interpenetrating POEGMA network. C2C12 myoblast proliferation in the PCL-POEGMA scaffolds was 50% higher than that observed on PCL-only scaffolds, a result attributed to the presence of the more hydrophilic POEGMA interpenetrating nanofiber network. Overall, this method is demonstrated to represent a facile single-step strategy to fabricate strong macroporous but still interfacially hydrophilic scaffolds for tissue engineering applications.

In situ-gelling hydrophobized starch nanoparticle-based nanoparticle network hydrogels for the effective delivery of intranasal olanzapine to treat brain disorders.

Intranasal (IN) delivery offers potential to deliver antipsychotic drugs with improved efficacy to the brain. However, the solubilization of such drugs and the frequency of required re-application both represent challenges to its practical implementation in treating various mental illnesses including schizophrenia. Herein, we report a sprayable nanoparticle network hydrogel (NNH) consisting of hydrophobically-modified starch nanoparticles (SNPs) and mucoadhesive chitosan oligosaccharide lactate (COL) that can gel in situ within the nasal cavity and release ultra-small penetrative SNPs over time. Hydrophobization of the SNPs enables enhanced uptake and prolonged release of poorly water soluble drugs such as olanzapine from the NNH depot through mucous and ultimately into the brain via the nose-to-brain (N2B) pathway. The hydrogel shows high in vitro cytocompatibility in mouse striatal neuron and human primary nasal cell lines and in vivo efficacy in an amphetamine-induced pre-clinical rat schizophrenia model, with IN-delivered NNH hydrogels maintaining successful attenuation of locomotor activity for up to 4 h while all other tested treatments (drug-only IN or conventional intraperitoneal delivery) failed to attenuate at any time point past 0.5 h. As such, in situ-gelling NNHs represent a safe excipient for the IN delivery of hydrophobic drugs directly to the brain using customized SNPs that exhibit high penetration and drug complexing properties to maximize effective drug delivery.

Supramolecularly-Cross-Linked Nanogel Assemblies for On-Demand, Ultrasound-Triggered Chemotherapy.

Stimulating the release of small nanoparticles (NPs) from a larger NP via the application of an exogenous stimulus offers the potential to address the different size requirements for circulation versus penetration that hinder chemotherapeutic drug delivery. Herein, we report a size-switching nanoassembly-based drug delivery system comprised of ultrasmall starch nanoparticles (SNPs, ∼20-50 nm major size fraction) encapsulated in a poly(oligo(ethylene glycol) methyl ether methacrylate) nanogel (POEGMA, ∼150 nm major size fraction) cross-linked via supramolecular PEG/α-cyclodextrin (α-CD) interactions. Upon heating the nanogel using a non-invasive, high-intensity focused ultrasound (HIFU) trigger, the thermoresponsive POEGMA-CD nanoassemblies are locally de-cross-linked, inducing in situ release of the highly penetrative drug-loaded SNPs. HIFU triggering increased the release of nanoassembly-loaded DOX from 17 to 37% after 3 h, a result correlated with significantly more effective tumor killing relative to nanoassemblies in the absence of HIFU or drug alone. Furthermore, 1.5× more total fluorescence was observed inside a tumor spheroid when nanoassemblies prepared with fluorophore-labeled SNPs were triggered with HIFU relative to the absence of HIFU. We anticipate this strategy holds promise for delivering tunable doses of chemotherapeutic drugs both at and within a tumor site using a non-invasive triggering approach.

The effect of comb length on the in vitro and in vivo properties of self-assembled poly(oligoethylene glycol methacrylate)-based block copolymer nanoparticles.

Comb copolymer analogues of poly(lactic acid)-polyethylene glycol block copolymers (PLA-b-PEG) offer potential to overcome the inherent chemistry and stability limitations of their linear block copolymer counterparts. Herein, we examine the differences between P(L)LA10K-b-PEG10K and linear-comb copolymer analogues thereof in which the linear PEG block is replaced by poly(oligo(ethylene glycol) methacrylate) (POEGMA) blocks with different side chain (comb) lengths but the same overall molecular weight. P(L)LA10K-b-POEGMA47510K and P(L)LA10K-b-POEGMA200010K block copolymers were synthesized via activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) and fabricated into self-assembled nanoparticles using flash nanoprecipitation via confined impinging jet mixing. Linear-comb copolymer analogues based on PLA-b-POEGMA yielded smaller but still well-controlled nanoparticle sizes (88 ± 2 nm and 114 ± 1 nm respectively compared to 159 ± 2 nm for P(L)LA10K-b-PEG10K nanoparticles) that exhibited improved colloidal stability relative to linear copolymer-based nanoparticles over a 15 day incubation period while maintaining comparably high cytocompatibility, although the comb copolymer analogues had somewhat lower loading capacity for doxorubicin hydrochloride. Cell spheroid studies showed that the linear-comb copolymers promoted enhanced tumor transport and thus cell killing compared to conventional linear block copolymers. In vivo studies showed all NP types could passively accumulate within implanted CT26 tumors but with different accumulation profiles, with P(L)LA10K-b-POEGMA200010K NPs showing continuous accumulation throughout the full 24 h monitoring period whereas tumor accumulation of P(L)LA10K-b-POEGMA47510K NPs was significant only between 8 h and 24 h. Overall, the linear-comb copolymer analogues exhibited superior stability, biodistribution, spheroid penetration, and inherent tunability over linear NP counterparts.

An Aptamer-Based Hydrogel Sensing Platform Enabling Low Micromolar Detection of Small Molecules Using Low Sample Volumes.

Poor fluorescence recovery at low analyte dosages and slow ligand binding kinetics are critical challenges currently limiting the use of aptamer-functionalized hydrogels for sensing small molecules. In this paper, we report an adenosine-responsive hydrogel sensor that integrates FRET-signaling aptamer switches into in situ-gelling thin-film hydrogels. The hydrogel sensor is able to entrap a high proportion of the sensing probes (>70% following vigorous washing), delay nucleolytic degradation, stabilize weak aptamer complexes to improve hybridization affinity and suppress fluorescence background, and provide high sensitivity in biological fluids (i.e., undiluted human serum). Furthermore, the developed hydrogel sensors were able to achieve low limits of detection (5.3 µM in buffer and 8.8 µM in serum) within 4 min of exposure to the sample, with signal generation requiring only 20 µL/well of analyte sample. The physical nature of the aptamer encapsulation allows this approach to accommodate virtually any small-molecule aptamer, avoiding the need for covalent anchoring and the complex modification of nucleic acid sequences typically required for effective aptamer-based molecular recognition.

Injectable and Dynamically Crosslinked Zwitterionic Hydrogels for Anti-Fouling and Tissue Regeneration Applications.

A zwitterionic injectable and degradable hydrogel based on hydrazide and aldehyde-functionalized [2-(methacryloyloxy)ethyl] dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS) precursor polymers that can address practical in vivo needs is reported. Zwitterion fusion interactions between the zwitterionic precursor polymers create a secondary physically crosslinked network to enable much more rapid gelation than previously reported with other synthetic polymers, facilitating rapid gelation at much lower polymer concentrations or degrees of functionalization than previously accessible in addition to promoting zero swelling and long-term degradation responses and significantly stiffer mechanics than are typically accessed with previously reported low-viscosity precursor gelation systems. The hydrogels maintain the highly anti-fouling properties of conventional zwitterionic hydrogels against proteins, mammalian cells, and bacteria while also promoting anti-fibrotic tissue responses in vivo. Furthermore, the use of the hydrogels for effective delivery and subsequent controlled release of viable cells with tunable profiles both in vitro and in vivo is demonstrated, including the delivery of myoblasts in a mouse skeletal muscle defect model for reducing the time between injury and functional mobility recovery. The combination of the injectability, degradability, and tissue compatibility achieved offers the potential to expand the utility of zwitterionic hydrogels in minimally invasive therapeutic applications.

A Nanocomposite Dynamic Covalent Cross-Linked Hydrogel Loaded with Fusidic Acid for Treating Antibiotic-Resistant Infected Wounds.

Methicillin-resistant Staphylococcus aureus (MRSA) is associated with high levels of morbidity and is considered a difficult-to-treat infection, often requiring nonstandard treatment regimens and antibiotics. Since over 40% of the emerging antibiotic compounds have insufficient solubility that limits their bioavailability and thus efficacy through oral or intravenous administration, it is crucial that alternative drug delivery products be developed for wound care applications. Existing effective treatments for soft tissue MRSA infections, such as fusidic acid (FA), which is typically administered orally, could also benefit from alternative routes of administration to improve local efficacy and bioavailability while reducing the required therapeutic dose. Herein, we report an antimicrobial poly(oligoethylene glycol methacrylate) (POEGMA)-based composite hydrogel loaded with fusidic acid-encapsulating self-assembled polylactic acid-b-poly(oligo(ethylene glycol) methyl ether methacrylate) (PLA-POEGMA) nanoparticles for the treatment of MRSA-infected skin wounds. The inclusion of the self-assembled nanoparticles (380 nm diameter when loaded with fusidic acid) does not alter the favorable mechanical properties and stability of the hydrogel in the context of its use as a wound dressing, while fusidic acid (FA) can be released from the hydrogel over ∼10 h via a diffusion-controlled mechanism. The antimicrobial studies demonstrate a clear zone of inhibition in vitro and a 1-2 order of magnitude inhibition of bacterial growth in vivo in an MRSA-infected full-thickness excisional murine wound model even at very low antibiotic doses. Our approach thus can both circumvent challenges in the local delivery of hydrophobic antimicrobial compounds and directly deliver antimicrobials into the wound to effectively combat methicillin-resistant infections using a fraction of the drug dose required using other clinically relevant strategies.

Injectable On-Demand Pulsatile Drug Delivery Hydrogels Using Alternating Magnetic Field-Triggered Polymer Glass Transitions.

Remote-controlled pulsatile or staged release has significant potential in a wide range of therapeutic treatments. However, most current approaches are hindered by the low resolution between the on- and off-states of drug release and the need for surgical implantation of larger controlled-release devices. Herein, we describe a method that addresses these limitations by combining injectable hydrogels, superparamagnetic iron oxide nanoparticles (SPIONs) that heat when exposed to an alternating magnetic field (AMF), and polymeric nanoparticles with a glass transition temperature (Tg) just above physiological temperature. Miniemulsion polymerization was used to fabricate poly(methyl methacrylate-co-butyl methacrylate) (p(MMA-co-BMA)) nanoparticles loaded with a model hydrophobic drug and tuned to have a Tg value just above physiological temperature (∼43 °C). Co-encapsulation of these drug-loaded nanoparticles with SPIONs inside a carbohydrate-based injectable hydrogel matrix (formed by rapid hydrazone cross-linking chemistry) enables injection and immobilization of the nanoparticles at the target site. Temperature cycling facilitated a 2.5:1 to 6:1 on/off rhodamine release ratio when the nanocomposites were switched between 37 and 45 °C; release was similarly enhanced by exposing the nanocomposite hydrogel to an AMF to drive heating, with enhanced release upon pulsing observed even 1 week after injection. Coupled with the apparent cytocompatibility of all of the nanocomposite components, these injectable nanocomposite hydrogels are promising as minimally invasive but remotely actuated release delivery vehicles capable of complex release kinetics with high on-off resolution.

Reactive Cell Electrospinning of Anisotropically Aligned and Bilayer Hydrogel Nanofiber Networks.

Structured hydrogels that incorporate aligned nanofibrous morphologies have been demonstrated to better replicate the structures of native extracellular matrices and thus their function in guiding cell responses. However, current techniques for nanofiber fabrication are limited in their ability to create hydrogel scaffolds with tunable directional alignments and cell types/densities, as required to reproduce more complex native tissue structures. Herein, we leverage a reactive cell electrospinning technique based on the dynamic covalent cross-linking of poly(ethylene glycol methacrylate (POEGMA) precursor polymers to fabricate aligned hydrogel nanofibers that can be directly loaded with cells during the electrospinning process. The scaffolds were found to support high C2C12 myoblast viabilities greater than 85% over 14 days, with changes in the electrospinning collector allowing for the single-step fabrication of nonaligned, aligned, or cross-aligned nanofibrous networks. Cell aspect ratios on aligned scaffolds were found on average to be 27% higher compared to those on nonaligned scaffolds; furthermore, cell-loaded bilayer scaffolds with perpendicular fiber alignments showed evidence of enabling localized directional cell responses to individual layer fiber directions while avoiding delamination between the layers. This fabrication approach thus offers potential for better mimicking the structure and thus function of aligned and multilayered tissues (e.g., smooth muscle, neural, or tendon tissues).

Overview of methods and considerations for the photodynamic inactivation of microorganisms for agricultural applications.

Antimicrobial resistance in agriculture is a global concern and carries huge financial consequences. Despite that, practical solutions for growers that are sustainable, low cost and environmentally friendly have been sparse. This has created opportunities for the agrochemical industry to develop pesticides with novel modes of action. Recently the use of photodynamic inactivation (PDI), classically used in cancer treatments, has been explored in agriculture as an alternative to traditional chemistries, mainly as a promising new approach for the eradication of pesticide resistant strains. However, applications in the field pose unique challenges and call for new methods of evaluation to adequately address issues specific to PDI applications in plants and challenges faced in the field. The aim of this review is to summarize in vitro, ex vivo, and in vivo/in planta experimental strategies and methods used to test and evaluate photodynamic agents as photo-responsive pesticides for applications in agriculture. The review highlights some of the strategies that have been explored to overcome challenges in the field.

Multicellular Layered Nanofibrous Poly(Oligo Ethylene Glycol Methacrylate) (POEGMA)-Based Hydrogel Scaffolds via Reactive Cell Electrospinning.

While hydrogels are demonstrated to be effective scaffolds for soft tissue engineering, existing fabrication techniques pose limitations in terms of being able to reproduce both the micro/nanofibrous structures of native extracellular matrix as well as the spatial arrangement of different cell types inherent of more complex tissues. Herein, a reactive cell electrospinning strategy is described using hydrazide and aldehyde-functionalized poly(oligoethylene glycol methacrylate) precursor polymers that can create nanofibrous hydrogel scaffolds with controllable local cell gradients using a sequential all-aqueous process that does not require additives or external energy. Cells can be encapsulated directly during the fabrication process in different layers within the scaffold, enabling localized segregation of different cell types within the structures without compromising their capacity to proliferate (≈4-fold increase in cell density over a 14 day incubation period). This sequential reactive electrospinning approach thus offers promise to generate coculture fibrous hydrogel networks in which both the nanoscale architecture and the cell distribution can be controlled, as it is essential to recreate more complex types of tissues.

Disulfide-Cross-Linked Nanogel-Based Nanoassemblies for Chemotherapeutic Drug Delivery.

Although nanoparticle-based chemotherapeutic strategies have gained in popularity, the efficacy of such therapies is still limited in part due to the different nanoparticle sizes needed to best accommodate different parts of the drug delivery pathway. Herein, we describe a nanogel-based nanoassembly based on the entrapment of ultrasmall starch nanoparticles (size 10-40 nm) within disulfide-crosslinked chondroitin sulfate-based nanogels (size 150-250 nm) to address this challenge. Upon exposure of the nanoassembly to the reductive tumor microenvironment, the chondroitin sulfate-based nanogel can degrade to release the doxorubicin-loaded starch nanoparticles in the tumor to facilitate improved intratumoral penetration. CT26 colon carcinoma spheroids could be efficiently penetrated by the nanoassembly (resulting in 1 order of magnitude higher DOX-derived fluorescence inside the spheroid relative to free DOX), while in vivo experiments showed that doxorubicin-loaded nanoassemblies reduced tumor sizes by 6× relative to saline controls and 2× relative to free DOX after 21 days. Together, these data suggest that nanogel-based nanoassemblies are a viable option for improving the efficacy and safety of nanoparticle-based drug delivery vehicles treating cancer.

Electrochemical DNAzyme-based biosensors for disease diagnosis.

DNAzyme-based electrochemical biosensors provide exceptional analytical sensitivity and high target recognition specificity for disease diagnosis. This review provides a critical perspective on the fundamental and applied impact of incorporating DNAzymes in the field of electrochemical biosensing. Specifically, we highlight recent advances in creating DNAzyme-based electrochemical biosensors for diagnosing infectious diseases, cancer and regulatory diseases. We also develop an understanding of challenges around translating the research in the field of DNAzyme-based electrochemical biosensors from labs to clinics, followed by a discussion on different strategies that can be applied to enhance the performance of the currently existing technologies to create truly point-of-care electrochemical DNAzyme biosensors.

Fabrication, Characterization and In Planta Uptake of Engineered Surfactant Nanovesicles for the Delivery of the Biostimulant Sodium Copper Chlorophyllin.

Effective delivery of agrochemicals requires control over bioactive release kinetics coupled with effective penetration of the bioactive into plants. Herein, we demonstrate the fabrication of hybrid nanovesicles based on sodium dodecylbenzenesulfonate (SDBS) and cetyltrimethylammonium bromide (CTAB) for enabling effective delivery of the biostimulant sodium copper chlorophyllin (Cu-chl) into plants. SDBS-CTAB nanovesicles exhibited a particle size of 107 nm with a well-defined spherical morphology, while modified formulations that included small fractions of the unsaturated dopant Span 80 yielded larger nanovesicles that were softer and more irregular in shape. All nanovesicles maintained high colloidal stability over >4 weeks and enabled sustained Cu-chl release, with the incorporation of Span 80 into the membranes enabling controllable acceleration of the release rate. Nanovesicle encapsulation improved the photostability of Cu-chl bioactive 3-4 × relative to that of free Cu-chl and enabled significant penetration of Cu-chl into the plant root without inducing any significant phytotoxicity.

FRESH Bioprinting of Dynamic Hydrazone-Cross-Linked Synthetic Hydrogels.

Dynamic covalent chemistry is an attractive cross-linking strategy for hydrogel bioinks due to its ability to mimic the dynamic interactions that are natively present in the extracellular matrix. However, the inherent challenges in mixing the reactive precursor polymers during printing and the tendency of the soft printed hydrogels to collapse during free-form printing have limited the use of such chemistry in 3D bioprinting cell scaffolds. Herein, we demonstrate 3D printing of hydrazone-cross-linked poly(oligoethylene glycol methacrylate) (POEGMA) hydrogels using the freeform reversible embedding of suspended hydrogels (FRESH) technique coupled with a customized low-cost extrusion bioprinter. The dynamic nature and reversibility of hydrazone cross-links enables reconfiguration of the initially more heterogeneous gel structure to form a more homogeneous internal gel structure, even for more highly cross-linked hydrogels, over a relatively short time (<3 days) while preserving the degradability of the scaffold over longer time frames. POEGMA hydrogels can successfully print NIH/3T3 fibroblasts and human umbilical vein endothelial cells while maintaining high cell viability (>80%) and supporting F-actin-mediated adhesion to the scaffold over a 14-day in vitro incubation period, demonstrating their potential use in practical tissue engineering applications.

The bending rigidity of the red blood cell cytoplasmic membrane.

An important mechanical property of cells is the membrane bending modulus, κ. In the case of red blood cells (RBCs) there is a composite membrane consisting of a cytoplasmic membrane and an underlying spectrin network. Literature values of κ are puzzling, as they are reported over a wide range, from 5 kBT to 230 kBT. To disentangle the contribution of the cytoplasmic membrane from the spectrin network, we investigated the bending of red blood cell cytoplasmic membranes (RBCcm) in the absence of spectrin and adenosine triphosphate (ATP). We used a combination of X-ray diffuse scattering (XDS), neutron spin-echo (NSE) spectrometry and Molecular Dynamics (MD) simulations. Our results indicate values of κ of order 4 kBT to 6 kBT, relatively small compared to literature values for most single component lipid bilayers. We suggest two ways this relative softness might confer biological advantage.

Using the Intranasal Route to Administer Drugs to Treat Neurological and Psychiatric Illnesses: Rationale, Successes, and Future Needs.

While the intranasal administration of drugs to the brain has been gaining both research attention and regulatory success over the past several years, key fundamental and translational challenges remain to fully leveraging the promise of this drug delivery pathway for improving the treatment of various neurological and psychiatric illnesses. In response, this review highlights the current state of understanding of the nose-to-brain drug delivery pathway and how both biological and clinical barriers to drug transport using the pathway can been addressed, as illustrated by demonstrations of how currently approved intranasal sprays leverage these pathways to enable the design of successful therapies. Moving forward, aiming to better exploit the understanding of this fundamental pathway, we also outline the development of nanoparticle systems that show improvement in delivering approved drugs to the brain and how engineered nanoparticle formulations could aid in breakthroughs in terms of delivering emerging drugs and therapeutics while avoiding systemic adverse effects.

Hydrogels for Tissue Engineering: Addressing Key Design Needs Toward Clinical Translation.

While the soft mechanics and tunable cell interactions facilitated by hydrogels have attracted significant interest in the development of functional hydrogel-based tissue engineering scaffolds, translating the many positive results observed in the lab into the clinic remains a slow process. In this review, we address the key design criteria in terms of the materials, crosslinkers, and fabrication techniques useful for fabricating translationally-relevant tissue engineering hydrogels, with particular attention to three emerging fabrication techniques that enable simultaneous scaffold fabrication and cell loading: 3D printing, in situ tissue engineering, and cell electrospinning. In particular, we emphasize strategies for manufacturing tissue engineering hydrogels in which both macroporous scaffold fabrication and cell loading can be conducted in a single manufacturing step - electrospinning, 3D printing, and in situ tissue engineering. We suggest that combining such integrated fabrication approaches with the lessons learned from previously successful translational experiences with other hydrogels represents a promising strategy to accelerate the implementation of hydrogels for tissue engineering in the clinic.

A DNA Barcode-Based Aptasensor Enables Rapid Testing of Porcine Epidemic Diarrhea Viruses in Swine Saliva Using Electrochemical Readout.

Pen-side testing of farm animals for infectious diseases is critical for preventing transmission in herds and providing timely intervention. However, most existing pathogen tests have to be conducted in centralized labs with sample-to-result times of 2-4 days. Herein we introduce a test that uses a dual-electrode electrochemical chip (DEE-Chip) and a barcode-releasing electroactive aptamer for rapid on-farm detection of porcine epidemic diarrhea viruses (PEDv). The sensor exploits inter-electrode spacing reduction and active field mediated transport to accelerate barcode movement from electroactive aptamers to the detection electrode, thus expediting assay operation. The test yielded a clinically relevant limit-of-detection of 6 nM (0.37 µg mL-1 ) in saliva-spiked PEDv samples. Clinical evaluation of this biosensor with 12 porcine saliva samples demonstrated a diagnostic sensitivity of 83 % and specificity of 100 % with a concordance value of 92 % at an analysis time of one hour.