Imaging in vivo acetylcholine release in the peripheral nervous system with a fluorescent nanosensor.

The ability to monitor the release of neurotransmitters during synaptic transmission would significantly impact the diagnosis and treatment of neurological diseases. Here, we present a DNA-based enzymatic nanosensor for quantitative detection of acetylcholine (ACh) in the peripheral nervous system of living mice. ACh nanosensors consist of DNA as a scaffold, acetylcholinesterase as a recognition component, pH-sensitive fluorophores as signal generators, and α-bungarotoxin as a targeting moiety. We demonstrate the utility of the nanosensors in the submandibular ganglia of living mice to sensitively detect ACh ranging from 0.228 to 358 µM. In addition, the sensor response upon electrical stimulation of the efferent nerve is dose dependent, reversible, and we observe a reduction of ∼76% in sensor signal upon pharmacological inhibition of ACh release. Equipped with an advanced imaging processing tool, we further spatially resolve ACh signal propagation on the tissue level. Our platform enables sensitive measurement and mapping of ACh transmission in the peripheral nervous system.

Clinicopathological associations of hemispheric dominance in primary progressive apraxia of speech.

OBJECTIVE: Primary progressive apraxia of speech (PPAOS) is associated with imaging abnormalities in the lateral premotor cortex (LPC) and supplementary motor area (SMA). It is not known whether greater involvement of these regions in either hemisphere is associated with demographics, presenting, and/or longitudinal features. METHODS: In 51 prospectively recruited PPAOS patients who completed [18 F]-fluorodeoxyglucose (FDG) positron emission tomography (PET), we classified patients as left-dominant, right-dominant, or symmetric, based on visual assessment of the LPC and SMA on FDG-PET. SPM and statistical analyses of regional metabolic values were performed. Diagnosis of PPAOS was made if apraxia of speech was present and aphasia absent. Thirteen patients completed ioflupane-123I (dopamine transporter [DAT]) scans. We compared cross-sectional and longitudinal clinicopathological, genetic, and neuroimaging characteristics across the three groups, with area under the receiver-operating curve (AUROC) determined as a measure of effect size. RESULTS: In all, 49% of the PPAOS patients were classified as left-dominant, 31% as right-dominant, and 20% as symmetric, which was supported by results from the SPM and regional analyses. There were no differences in baseline characteristics. Longitudinally, right-dominant PPAOS showed faster rates of progression of ideomotor apraxia (AUROC 0.79), behavioral disturbances (AUROC 0.84), including disinhibition symptoms (AUROC 0.82) and negative behaviors (AUROC 0.82), and parkinsonism (AUROC 0.75) compared to left-dominant PPAOS. Symmetric PPAOS showed faster rates of dysarthria progression compared to left-dominant (AUROC 0.89) and right-dominant PPAOS (AUROC 0.79). Five patients showed abnormal DAT uptake. Braak neurofibrillary tangle stage differed across groups (p = 0.01). CONCLUSIONS: Patients with PPAOS and a right-dominant pattern of hypometabolism on FDG-PET have the fastest rates of decline of behavioral and motor features.

Real-time particle-by-particle detection of erythrocyte-camouflaged microsensor with extended circulation time in the bloodstream.

Personalized medicine offers great potential benefits for disease management but requires continuous monitoring of drugs and drug targets. For instance, the therapeutic window for lithium therapy of bipolar disorder is very narrow, and more frequent monitoring of sodium levels could avoid toxicity. In this work, we developed and validated a platform for long-term, continuous monitoring of systemic analyte concentrations in vivo. First, we developed sodium microsensors that circulate directly in the bloodstream. We used "red blood cell mimicry" to achieve long sensor circulation times of up to 2 wk, while being stable, reversible, and sensitive to sodium over physiologically relevant concentration ranges. Second, we developed an external optical reader to detect and quantify the fluorescence activity of the sensors directly in circulation without having to draw blood samples and correlate the measurement with a phantom calibration curve to measure in vivo sodium. The reader design is inherently scalable to larger limbs, species, and potentially even humans. In combination, this platform represents a paradigm for in vivo drug monitoring that we anticipate will have many applications in the future.

Click Chemistry-Enabled Conjugation Strategy for Producing Dibenzodiazepinone-Type Fluorescent Probes To Target M2 Acetylcholine Receptors.

The development of fluorescently labeled receptor-targeting compounds represents a powerful pharmacological tool to study and characterize ligand-receptor interactions. Despite significant advances in developing sub-type-specific antagonists for muscarinic acetylcholine receptors (mAChRs), reports on antagonists feasible for click chemistry are less common. Here, we designed and synthesized an antagonist suitable for probe attachment through click chemistry, namely, dibenzodiazepinone (DIBA)-alkyne, based on a previously reported DIBA scaffold with a high binding affinity to type-2 mAChR (M2R). To demonstrate the versatility of DIBA-alkyne as a building block for bioconjugates, we assembled DIBA-alkyne with Cyanine5 fluorophores (Cy5) and polyethylene glycol (PEG) biomolecules to obtain fluorescent DIBA antagonist (DIBA-Cy5) and fluorescent DIBA PEG derivatives. Flow cytometric analysis showed that DIBA-Cy5 possessed a high binding affinity to M2R (Kd = 1.80 nM), a two-order magnitude higher binding affinity than M1R. Fluorescent DIBA PEG derivatives maintained a potent binding to the M2R (Kd ≤ 4 nM), confirmed by confocal microscopic imaging. Additionally, DIBA-Cy5 can serve as a fluorescent ligand in the receptor-ligand competitive binding assay for other mAChR ligands, an attractive alternative to the traditional radioligand-based assay. The competitive binding mode between DIBA-Cy5 and orthosteric antagonist atropine/allosteric modulator LY2119620 indicated a dualsteric binding mode of the DIBA-type antagonist to M2R. Lastly, we demonstrated the direct staining of DIBA-Cy5 to M2R receptors in the sinoatrial node of a mouse heart. The adaptability of the clickable DIBA antagonist to a wide range of fluorophores and biomolecules can facilitate its use in various biomedical applications such as binding assays that screen compounds for M2R as the receptor target.

Gadolinium-based MRI contrast agent for the detection of tyrosinase.

Tyrosinase is a key enzyme that has long been considered as a biomarker for melanoma as it catalyzes the oxidation of tyrosine and l-DOPA in melanogenesis. Recent studies also suggest a link between tyrosinase activity and Parkinson's disease; however, the mechanism of tyrosinase-mediated melanin formation in the brain is poorly understood. To better understand this connection, more advanced tools for the detection of tyrosinase in the brain are required. Herein, we successfully designed and synthesized a tyrosinase-targeting Gd(iii)-based MR contrast agent Tyr-GBCA 1. Tyr-GBCA 1 was synthesized by linking m-hydroxyphenyl to Gd-DOTA via a self-immolative linker. Tyr-GBCA 1 shows a 21% increase in the T1 relaxation rate (R1) in the presence of tyrosinase in artificial cerebral spinal fluid. Furthermore, Tyr-GBCA 1 is unreactive to hydrogen peroxide, which is a potential interferent in oxidation-based tyrosinase sensing systems. The reaction mechanism of the probe was studied by electrospray ionization (ESI) mass spectrometry and supports the cleavage of a reaction site.

Optical Probes for Neurobiological Sensing and Imaging.

Fluorescent nanosensors and molecular probes are next-generation tools for imaging chemical signaling inside and between cells. Electrophysiology has long been considered the gold standard in elucidating neural dynamics with high temporal resolution and precision, particularly on the single-cell level. However, electrode-based techniques face challenges in illuminating the specific chemicals involved in neural cell activation with adequate spatial information. Measuring chemical dynamics is of fundamental importance to better understand synergistic interactions between neurons as well as interactions between neurons and non-neuronal cells. Over the past decade, significant technological advances in optical probes and imaging methods have enabled entirely new possibilities for studying neural cells and circuits at the chemical level. These optical imaging modalities have shown promise for combining chemical, temporal, and spatial information. This potential makes them ideal candidates to unravel the complex neural interactions at multiple scales in the brain, which could be complemented by traditional electrophysiological methods to obtain a full spatiotemporal picture of neurochemical dynamics. Despite the potential, only a handful of probe candidates have been utilized to provide detailed chemical information in the brain. To date, most live imaging and chemical mapping studies rely on fluorescent molecular indicators to report intracellular calcium (Ca2+) dynamics, which correlates with neuronal activity. Methodological advances for monitoring a full array of chemicals in the brain with improved spatial, temporal, and chemical resolution will thus enable mapping of neurochemical circuits with finer precision. On the basis of numerous studies in this exciting field, we review the current efforts to develop and apply a palette of optical probes and nanosensors for chemical sensing in the brain. There is a strong impetus to further develop technologies capable of probing entire neurobiological units with high spatiotemporal resolution. Thus, we introduce selected applications for ion and neurotransmitter detection to investigate both neurons and non-neuronal brain cells. We focus on families of optical probes because of their ability to sense a wide array of molecules and convey spatial information with minimal damage to tissue. We start with a discussion of currently available molecular probes, highlight recent advances in genetically modified fluorescent probes for ions and small molecules, and end with the latest research in nanosensors for biological imaging. Customizable, nanoscale optical sensors that accurately and dynamically monitor the local environment with high spatiotemporal resolution could lead to not only new insights into the function of all cell types but also a broader understanding of how diverse neural signaling systems act in conjunction with neighboring cells in a spatially relevant manner.

Comparative Study of Poly (ε-Caprolactone) and Poly(Lactic-co-Glycolic Acid) -Based Nanofiber Scaffolds for pH-Sensing.

PURPOSE: This study aims to develop biodegradable and biocompatible polymer-based nanofibers that continuously monitor pH within microenvironments of cultured cells in real-time. In the future, these fibers will provide a scaffold for tissue growth while simultaneously monitoring the extracellular environment. METHODS: Sensors to monitor pH were created by directly electrospinning the sensor components within a polymeric matrix. Specifically, the entire fiber structure is composed of the optical equivalent of an electrode, a pH-sensitive fluorophore, an ionic additive, a plasticizer, and a polymer to impart mechanical stability. The resulting poly(ε-caprolactone) (PCL) and poly(lactic-co-glycolic acid) (PLGA) based sensors were characterized by morphology, dynamic range, reversibility and stability. Since PCL-based nanofibers delivered the most desirable analytical response, this matrix was used for cellular studies. RESULTS: Electrospun nanofiber scaffolds (NFSs) were created directly out of optode material. The resulting NFS sensors respond to pH changes with a dynamic range centered at 7.8 ± 0.1 and 9.6 ± 0.2, for PCL and PLGA respectively. NFSs exhibited multiple cycles of reversibility with a lifetime of at least 15 days with preservation of response characteristics. By comparing the two NFSs, we found PCL-NFSs are more suitable for pH sensing due to their dynamic range and superior reversibility. CONCLUSION: The proposed sensing platform successfully exhibits a response to pH and compatibility with cultured cells. NSFs will be a useful tool for creating 3D cellular scaffolds that can monitor the cellular environment with applications in fields such as drug discovery and tissue engineering.

Development of an Optical Nanosensor Incorporating a pH-Sensitive Quencher Dye for Potassium Imaging.

One of the key challenges in the design of a sensor for measuring extracellular changes in potassium concentration is selectivity against the competing cation, sodium. Here, we present an optode-based nanosensor selective to potassium ions, owing to the addition of a pH-sensitive quencher molecule paired with a static fluorophore. The nanosensor was fabricated using emulsification and characterized in solution by absorbance and fluorescence spectroscopy. The resulting nanosensor detected potassium with nearly 1 order of magnitude higher selectivity compared to our chromoionophore-based optode nanosensors. In addition to the improved selectivity, the nanosensor has the following properties required for measurements in a biological environment: (1) a physiologically relevant dynamic range, (2) response to potassium ions at a physiological ionic strength, and (3) response to serum potassium in the presence of fouling biological components. The potassium nanosensor described in this study is envisioned to have application in cellular imaging and drug screening.

Glucose-sensitive nanofiber scaffolds with an improved sensing design for physiological conditions.

Continuous physiological monitoring of electrolytes and small molecules such as glucose, creatinine, and urea is currently unavailable but achieving such a capability would be a major milestone for personalized medicine. Optode-based nanosensors are an appealing analytical platform for designing in vivo monitoring systems. In addition to the necessary analytical performance, such nanosensors must also be biocompatible and remain immobile at the implantation site. Blood glucose in particular remains a difficult but high-value analyte to monitor continuously. Previously, we developed glucose-sensitive nanosensors that measure glucose by a competitive binding mechanism between glucose and a fluorescent dye to 4-carboxy-3-fluorophenyl boronic acid. To improve the sensitivity and residency time of our reported sensors, we present here a series of new derivatives of 4-carboxy-3-fluorophenyl boronic acid that we screened in a macrosensor format before translating into a nanofiber format with electrospinning. The lead candidate was then implanted subdermally and its residency time was compared to spherical nanosensor analogues. The nanofiber scaffolds were markedly more stable at the implantation site whereas spherical nanosensors diffused away within three hours. Based on the enhanced sensitivity of the new boronic acids and the residency time of nanofibers, this sensor configuration is an important step towards continuous monitoring of glucose and other analytes.

Quadruplex Integrated DNA (QuID) Nanosensors for Monitoring Dopamine.

Dopamine is widely innervated throughout the brain and critical for many cognitive and motor functions. Imbalances or loss in dopamine transmission underlie various psychiatric disorders and degenerative diseases. Research involving cellular studies and disease states would benefit from a tool for measuring dopamine transmission. Here we show a Quadruplex Integrated DNA (QuID) nanosensor platform for selective and dynamic detection of dopamine. This nanosensor exploits DNA technology and enzyme recognition systems to optically image dopamine levels. The DNA quadruplex architecture is designed to be compatible in physically constrained environments (110 nm) with high flexibility, homogeneity, and a lower detection limit of 110 µM.

Implantable nanosensors: toward continuous physiologic monitoring.

Continuous physiologic monitoring would add greatly to both home and clinical medical treatment for chronic conditions. Implantable nanosensors are a promising platform for designing continuous monitoring systems. This Feature reviews design considerations and current approaches toward such devices.

Fluorescent sensors for the basic metabolic panel enable measurement with a smart phone device over the physiological range.

The advanced functionality of portable devices such as smart phones provides the necessary hardware to potentially perform complex diagnostic measurements in any setting. Recent research and development have utilized cameras and data acquisition properties of smart phones to create diagnostic approaches for a variety of diseases or pollutants. However, in concentration measurements, such as blood glucose, the performance of handheld diagnostic devices depends largely on the sensing mechanism. To expand measurements to multiple components, often necessary in medical tests, with a single diagnostic device, robust platform based sensors are needed. Here, we developed a suite of dual wavelength fluorescent sensors with response characteristics necessary to measure each component of a basic metabolic panel, a common clinical measurement. Furthermore, the response of these sensors could be measured with a simple optical setup to convert a smart phone into a fluorescence measurement instrument. This approach could be used as a mobile basic metabolic panel measurement system for point of care diagnostics.

Microworm optode sensors limit particle diffusion to enable in vivo measurements.

There have been a variety of nanoparticles created for in vivo uses ranging from gene and drug delivery to tumor imaging and physiological monitoring. The use of nanoparticles to measure physiological conditions while being fluorescently addressed through the skin provides an ideal method toward minimally invasive health monitoring. Here we create unique particles that have all the necessary physical characteristics to serve as in vivo reporters, but with minimized diffusion from the point of injection. These particles, called microworms, have a cylindrical shape coated with a biocompatible porous membrane that possesses a large surface-area-to-volume ratio while maintaining a large hydrodynamic radius. We use these microworms to create fluorescent sodium sensors for use as in vivo sodium concentration detectors after subcutaneous injection. However, the microworm concept has the potential to extend to the immobilization of other types of polymers for continuous physiological detection or delivery of molecules.

pH-responsive i-motif-conjugated nanoparticles for MRI analysis.

Gadolinium (Gd)-based contrast agents (CAs) are widely used to enhance anatomical details in magnetic resonance imaging (MRI). Significant research has expanded the field of CAs into bioresponsive CAs by modulating the signal to image and monitor biochemical processes, such as pH. In this work, we introduce the modular, dynamic actuation mechanism of DNA-based nanostructures as a new way to modulate the MRI signal based on the rotational correlation time, τR. We combined a pH-responsive oligonucleotide (i-motif) and a clinical standard CA (Gd-DOTA) to develop a pH-responsive MRI CA. The i-motif folds into a quadruplex under acidic conditions and was incorporated onto gold nanoparticles (iM-GNP) to achieve increased relaxivity, r1, compared to the unbound i-motif. In vitro, iM-GNP resulted in a significant increase in r1 over a decreasing pH range (7.5-4.5) with a calculated pKa = 5.88 ± 0.01 and a 16.7% change per 0.1 pH unit. In comparison, a control CA with a non-responsive DNA strand (T33-GNP) did not show a significant change in r1 over the same pH range. The iM-GNP was further evaluated in 20% human serum and demonstrated a 28.14 ± 11.2% increase in signal from neutral pH to acidic pH. This approach paves a path for novel programmable, dynamic DNA-based complexes for τR-modulated bioresponsive MRI CAs.

Phosphorescent nanosensors for in vivo tracking of histamine levels.

Continuously tracking bioanalytes in vivo will enable clinicians and researchers to profile normal physiology and monitor diseased states. Current in vivo monitoring system designs are limited by invasive implantation procedures and biofouling, limiting the utility of these tools for obtaining physiologic data. In this work, we demonstrate the first success in optically tracking histamine levels in vivo using a modular, injectable sensing platform based on diamine oxidase and a phosphorescent oxygen nanosensor. Our new approach increases the range of measurable analytes by combining an enzymatic recognition element with a reversible nanosensor capable of measuring the effects of enzymatic activity. We use these enzyme nanosensors (EnzNS) to monitor the in vivo histamine dynamics as the concentration rapidly increases and decreases due to administration and clearance. The EnzNS system measured kinetics that match those reported from ex vivo measurements. This work establishes a modular approach to in vivo nanosensor design for measuring a broad range of potential target analytes. Simply replacing the recognition enzyme, or both the enzyme and nanosensor, can produce a new sensor system capable of measuring a wide range of specific analytical targets in vivo.

Nucleic acid nanostructures for in vivo applications: The influence of morphology on biological fate.

The development of programmable biomaterials for use in nanofabrication represents a major advance for the future of biomedicine and diagnostics. Recent advances in structural nanotechnology using nucleic acids have resulted in dramatic progress in our understanding of nucleic acid-based nanostructures (NANs) for use in biological applications. As the NANs become more architecturally and functionally diverse to accommodate introduction into living systems, there is a need to understand how critical design features can be controlled to impart desired performance in vivo. In this review, we survey the range of nucleic acid materials utilized as structural building blocks (DNA, RNA, and xenonucleic acids), the diversity of geometries for nanofabrication, and the strategies to functionalize these complexes. We include an assessment of the available and emerging characterization tools used to evaluate the physical, mechanical, physiochemical, and biological properties of NANs in vitro. Finally, the current understanding of the obstacles encountered along the in vivo journey is contextualized to demonstrate how morphological features of NANs influence their biological fates. We envision that this summary will aid researchers in the designing novel NAN morphologies, guide characterization efforts, and design of experiments and spark interdisciplinary collaborations to fuel advancements in programmable platforms for biological applications.

Enhancing Biocidal Capability in Cuprite Coatings.

The SARS-CoV-2 global pandemic has reinvigorated interest in the creation and widespread deployment of durable, cost-effective, and environmentally benign antipathogenic coatings for high-touch public surfaces. While the contact-kill capability and mechanism of metallic copper and its alloys are well established, the biocidal activity of the refractory oxide forms remains poorly understood. In this study, commercial cuprous oxide (Cu2O, cuprite) powder was rapidly nanostructured using high-energy cryomechanical processing. Coatings made from these processed powders demonstrated a passive "contact-kill" response to Escherichia coli (E. coli) bacteria that was 4× (400%) faster than coatings made from unprocessed powder. No viable bacteria (>99.999% (5-log10) reduction) were detected in bioassays performed after two hours of exposure of E. coli to coatings of processed cuprous oxide, while a greater than 99% bacterial reduction was achieved within 30 min of exposure. Further, these coatings were hydrophobic and no external energy input was required to activate their contact-kill capability. The upregulated antibacterial response of the processed powders is positively correlated with extensive induced crystallographic disorder and microstrain in the Cu2O lattice accompanied by color changes that are consistent with an increased semiconducting bandgap energy. It is deduced that cryomilling creates well-crystallized nanoscale regions enmeshed within the highly lattice-defective particle matrix. Increasing the relative proportion of lattice-defective cuprous oxide exposed to the environment at the coating surface is anticipated to further enhance the antipathogenic capability of this abundant, inexpensive, robust, and easily handled material for wider application in contact-kill surfaces.

Biodegradable optode-based nanosensors for in vivo monitoring.

Optode-based fluorescent nanosensors are being developed for monitoring important disease states such as hyponatremia and diabetes. However, traditional optode-based sensors are composed of nonbiodegradable polymers such as poly(vinyl chloride) (PVC) raising toxicity concerns for long-term in vivo use. Here, we report the development of the first biodegradable optode-based nanosensors that maintain sensing characteristics similar to those of traditional optode sensors. The polymer matrix of these sensors is composed of polycaprolactone (PCL) and a citric acid ester plasticizer. The PCL-based nanosensors yielded a dynamic and reversible response to sodium, were tuned to respond to extracellular sodium concentrations, and had a lifetime of at least 14 days at physiological temperature. When in the presence of lipase, the nanosensors degraded within 4 h at lipase concentrations found in the liver but were present after 3 days at lipase concentrations found in serum. The development of biodegradable nanosensors is not only a positive step towards their future use in in vivo applications, but they also represent a new sensor platform that can be extended to other sensing mechanisms.

Visualizing sodium dynamics in isolated cardiomyocytes using fluorescent nanosensors.

Regulation of sodium flux across the cell membrane plays a vital role in the generation of action potentials and regulation of membrane excitability in cells such as cardiomyocytes and neurons. Alteration of sodium channel function has been implicated in diseases such as epilepsy, long QT syndrome, and heart failure. However, single cell imaging of sodium dynamics has been limited due to the narrow selection of fluorescent sodium indicators available to researchers. Here we report, the detection of spatially defined sodium activity during action potentials. Fluorescent nanosensors that measure sodium in real-time, are reversible and are completely selective over other cations such as potassium that were used to image sodium. The use of the nanosensors in vitro was validated by determining drug-induced activation in heterologous cells transfected with the voltage-gated sodium channel Na(V)1.7. Spatial information of sodium concentrations during action potentials will provide insight at the cellular level on the role of sodium and how slight changes in sodium channel function can affect the entirety of an action potential.

In vivo histamine optical nanosensors.

In this communication we discuss the development of ionophore based nanosensors for the detection and monitoring of histamine levels in vivo. This approach is based on the use of an amine-reactive, broad spectrum ionophore which is capable of recognizing and binding to histamine. We pair this ionophore with our already established nanosensor platform, and demonstrate in vitro and in vivo monitoring of histamine levels. This approach enables capturing rapid kinetics of histamine after injection, which are more difficult to measure with standard approaches such as blood sampling, especially on small research models. The coupling together of in vivo nanosensors with ionophores such as nonactin provide a way to generate nanosensors for novel targets without the difficult process of designing and synthesizing novel ionophores.