Surface Acoustic Wave-Driven Enhancement of Enzyme-Linked Immunosorbent Assays: ELISAW.

Enzyme-linked immunosorbent assays (ELISAs) are widely used in biology and clinical diagnosis. Relying on antigen-antibody interaction through diffusion, the standard ELISA protocol can be time-consuming, preventing its use in rapid diagnostics. We present a time-saving and more sensitive ELISA without changing the standard setup and protocol, using surface acoustic waves (SAWs) to enhance performance. Each step of the assay, from the initial antibody binding onto the walls of the well plate to the target analyte molecules' binding for detection─except, notably, for the blocking step─is improved principally via acoustic streaming-driven advection. Using SAWs, the time required for one step of an example ELISA is reduced from 60 to 15 min to achieve the same binding amount. By extending the duration of SAW exposure to 20 min, the sensitivity can be significantly improved over the 60 min, 35 °C ELISA without SAWs. It is also possible to confer beneficial improvements to bead-based ELISA by combining it with SAWs to further reduce the time required for binding to 2 min. By significantly increasing the speed of ELISA, its utility may be improved for a wide range of point-of-care diagnostics applications.

Application of Statistical Analysis and Machine Learning to Identify Infants' Abnormal Suckling Behavior.

OBJECTIVE: Identify infants with abnormal suckling behavior from simple non-nutritive suckling devices. BACKGROUND: While it is well known breastfeeding is beneficial to the health of both mothers and infants, breastfeeding ceases in 75 percent of mother-child dyads by 6 months. The current standard of care lacks objective measurements to screen infant suckling abnormalities within the first few days of life, a critical time to establish milk supply and successful breastfeeding practices. MATERIALS AND METHODS: A non-nutritive suckling vacuum measurement system, previously developed by the authors, is used to gather data from 91 healthy full-term infants under thirty days old. Non-nutritive suckling was recorded for a duration of sixty seconds. We establish normative data for the mean suck vacuum, maximum suck vacuum, suckling frequency, burst duration, sucks per burst, and vacuum signal shape. We then apply computational methods (Mahalanobis distance, KNN) to detect anomalies in the data to identify infants with abnormal suckling. We finally provide case studies of healthy newborn infants and infants diagnosed with ankyloglossia. RESULTS: In a series of case evaluations, we demonstrate the ability to detect abnormal suckling behavior using statistical analysis and machine learning. We evaluate cases of ankyloglossia to determine how oral dysfunction and surgical interventions affect non-nutritive suckling measurements. CONCLUSIONS: Statistical analysis (Mahalanobis Distance) and machine learning [K nearest neighbor (KNN)] can be viable approaches to rapidly interpret infant suckling measurements. Particularly in practices using the digital suck assessment with a gloved finger, it can provide a more objective, early stage screening method to identify abnormal infant suckling vacuum. This approach for identifying those at risk for breastfeeding complications is crucial to complement complex emerging clinical evaluation technology. CLINICAL IMPACT: By analyzing non-nutritive suckling using computational methods, we demonstrate the ability to detect abnormal and normal behavior in infant suckling that can inform breastfeeding intervention pathways in clinic.Clinical and Translational Impact Statement: The work serves to shed light on the lack of consensus for determining appropriate intervention pathways for infant oral dysfunction. We demonstrate using statistical analysis and machine learning that normal and abnormal infant suckling can be identified and used in determining if surgical intervention is a necessary solution to resolve infant feeding difficulties.

Extracellular vesicles-based point-of-care testing for the diagnosis and monitoring of Alzheimer's disease.

Alzheimer's disease (AD) is a debilitating condition that affects millions of people worldwide. One promising strategy for detecting and monitoring AD early on is using extracellular vesicles (EVs)-based point-of-care testing; however, diagnosing AD using EVs poses a challenge due to the low abundance of EV-biomarkers. Here, we present a fully integrated organic electrochemical transistor (OECT) that enables high accuracy, speed, and convenience in the detection of EVs from AD patients. We incorporated self-aligned acoustoelectric enhancement of EVs on a chip that rapidly propels, enriches, and specifically binds EVs to the OECT detection area. With our enhancement of pre-concentration, we increased the sensitivity to a limit of detection of 500 EV particles/µL and reduced the required detection time to just two minutes. We also tested the sensor on an AD mouse model to monitor AD progression, examined mouse Aß EVs at different time courses, and compared them with intraneuronal Aß cumulation using MRI. This innovative technology has the potential to diagnose Alzheimer's and other neurodegenerative diseases accurately and quickly, enabling monitoring of disease progression and treatment response.

Glycocalyx transduces membrane leak in brain tumor cells exposed to sharp magnetic pulsing.

Mechanisms by which electric (E) or magnetic (B) fields might be harnessed to affect tumor cell behavior remain poorly defined, presenting a barrier to translation. We hypothesized in early studies that the glycocalyx of lung cancer cells might play a role in mediating plasma membrane leak by low-frequency pulsed magnetic fields (Lf-PMF) generated on a low-energy solenoid platform. In testing glioblastoma and neuroblastoma cells known to overexpress glycoproteins rich in modifications by the anionic glycan sialic acid (Sia), exposure of brain tumor cells on the same platform to a pulse train that included a 5 min 50Hz Lf-PMF (dB/dt ∼ 2 T/s at 10 ms pulse widths) induced a very modest but significant protease leak above that of control nonexposed cells (with modest but significant reductions in long-term tumor cell viability after the 5 min exposure). Using a markedly higher dB/dt system (80 T/s pulses, 70 µs pulse-width at 5.9 cm from a MagVenture coil source) induced markedly greater leak by the same cells, and eliminating Sia by treating cells with AUS sialidase immediately preexposure abrogated the effect entirely in SH-SY5Y neuroblastoma cells, and partially in T98G glioblastoma cells. The system demonstrated significant leak (including inward leak of propidium iodide), with reduced leak at lower dB/dt in a variety of tumor cells. The ability to abrogate Lf-PMF protease leak by pretreatment with sialidase in SH-SY5Y brain tumor cells or with heparin lyase in A549 lung tumor cells indicated the importance of heavy Sia or heparan sulfate glycosaminoglycan glycocalyx modifications as dominant glycan species mediating Lf-PMF membrane leak in respective tumor cells. This "first-physical" Lf-PMF tumor glycocalyx event, with downstream cell stress, may represent a critical and "tunable" transduction mechanism that depends on characteristic anionic glycans overexpressed by distinct malignant tumors.

Onset of Visible Capillary Waves from High-Frequency Acoustic Excitation.

Remarkably, the interface of a fluid droplet will produce visible capillary waves when exposed to acoustic waves. For example, a small (∼1 µL) sessile droplet will oscillate at a low ∼102 Hz frequency when weakly driven by acoustic waves at ∼106 Hz frequency and beyond. We measured such a droplet's interfacial response to 6.6 MHz ultrasound to gain insight into the energy transfer mechanism that spans these vastly different time scales, using high-speed microscopic digital transmission holography, a unique method to capture three-dimensional surface dynamics at nanometer space and microsecond time resolutions. We show that low-frequency capillary waves are driven into existence via a feedback mechanism between the acoustic radiation pressure and the evolving shape of the fluid interface. The acoustic pressure is distributed in the standing wave cavity of the droplet, and as the shape of the fluid interface changes in response to the distributed pressure present on the interface, the standing wave field also changes shape, feeding back to produce changes in the acoustic radiation pressure distribution in the cavity. A physical model explicitly based upon this proposed mechanism is provided, and simulations using it were verified against direct observations of both the microscale droplet interface dynamics from holography and internal pressure distributions using microparticle image velocimetry. The pressure-interface feedback model accurately predicts the vibration amplitude threshold at which capillary waves appear, the subsequent amplitude and frequency of the capillary waves, and the distribution of the standing wave pressure field within the sessile droplet responsible for the capillary waves.

Non-Nutritive Suckling System for Real-Time Characterization of Intraoral Vacuum Profile in Full Term Neonates.

Infant breastfeeding diagnostics remain subjective due to the absence of instrumentation to objectively measure and understand infant oral motor skills and suckling characteristics. Qualitative diagnostic exams, such as the digital suck assessment which relies upon a clinician's gloved finger inserted into the infant's mouth, produce a diversity of diagnoses and intervention pathways due to their subjective nature. In this paper, we report on the design of a non-nutritive suckling (NNS) system which quantifies and analyzes quantitative intraoral vacuum and sucking patterns of full-term neonates in real time. In our study, we evaluate thirty neonate suckling profiles to demonstrate the technical and clinical feasibility of the system. We successfully extract the mean suck vacuum, maximum suck vacuum, frequency, burst duration, number of sucks per burst, number of sucks per minute, and number of bursts per minute. In addition, we highlight the discovery of three intraoral vacuum profile shapes that are found to be correlated to different levels of suckling characteristics. These results establish a framework for future studies to evaluate oromotor dysfunction that affect the appearance of these signals based on established normal profiles. Ultimately, with the ability to easily and quickly capture intraoral vacuum data, clinicians can more accurately perform suckling assessments to provide timely intervention and assist mothers and infants towards successful breastfeeding outcomes.

Modeling fast acoustic streaming: Steady-state and transient flow solutions.

Traditionally, acoustic streaming is assumed to be a steady-state, relatively slow fluid response to passing acoustic waves. This assumption, the so-called slow streaming assumption, was made over a century ago by Lord Rayleigh. It produces a tractable asymptotic perturbation analysis from the nonlinear governing equations, separating the acoustic field from the acoustic streaming that it generates. Unfortunately, this assumption is often invalid in the modern microacoustofluidics context, where the fluid flow and acoustic particle velocities are comparable. Despite this issue, the assumption is still widely used today, as there is no suitable alternative. We describe a mathematical method to supplant the classic approach and properly treat the spatiotemporal scale disparities present between the acoustics and remaining fluid dynamics. The method is applied in this work to well-known problems of semi-infinite extent defined by the Navier-Stokes equations, and preserves unsteady fluid behavior driven by the acoustic wave. The separation of the governing equations between the fast (acoustic) and slow (hydrodynamic) spatiotemporal scales are shown to naturally arise from the intrinsic properties of the fluid under forcing, not by arbitrary assumption beforehand. Solution of the unsteady streaming field equations provides physical insight into observed temporal evolution of bulk streaming flows that, to date, have not been modeled. A Burgers equation is derived from our method to represent unsteady flow. By then assuming steady flow, a Riccati equation is found to represent it. Solving these equations produces direct, concise insight into the nonlinearity of the acoustic streaming phenomenon alongside an absolute, universal upper bound of 50% for the energy efficiency in transducing acoustic energy input to the acoustic streaming energy output. Rigorous validation with respect to experimental and theoretical results from the classic literature is presented to connect this work to past efforts by many authors.

Quantifying cell adhesion through forces generated by acoustic streaming.

The strength of cell adhesion is important in understanding the cell's health and in culturing them. Quantitative measurement of cell adhesion strength is a significant challenge in bioengineering research. For this, the present study describes a system that can measure cell adhesion strength using acoustic streaming induced by Lamb waves. Cells are cultured on an ultrasound transducer using a range of preculture and incubation times with phosphate-buffered saline (PBS) just before the measurement. Acoustic streaming is then induced using several Lamb wave intensities, exposing the cells to shear flows and eventually detaching them. By relying upon a median detachment rate of 50 %, the corresponding detachment force, or force of cell adhesion, was determined to be on the order of several nN, consistent with previous reports. The stronger the induced shear flow, the more cells were detached. Further, we employed a preculture time of 8 to 24 h and a PBS incubation time of 0 to 60 min, producing cell adhesion forces that varied from 1.2 to 13 nN. Hence, the developed system can quantify cell adhesion strength over a wide range, possibly offering a fundamental tool for cell-based bioengineering.

Acousto-Photolithography for Programmable Shape Deformation of Composite Hydrogel Sheets.

Stimuli-responsive hydrogels with programmable shapes produced by defined patterns of particles are of great interest for the fabrication of small-scale soft actuators and robots. Patterning the particles in the hydrogels during fabrication generally requires external magnetic or electric fields, thus limiting the material choice for the particles. Acoustically driven particle manipulation, however, solely depends on the acoustic impedance difference between the particles and the surrounding fluid, making it a more versatile method to spatially control particles. Here, an approach is reported by combining direct acoustic force to align photothermal particles and photolithography to spatially immobilize these alignments within a temperature-responsive poly(N-isopropylacrylamide) hydrogel to trigger shape deformation under temperature change and light exposure. The spatial distribution of particles can be tuned by the power and frequency of the acoustic waves. Specifically, changing the spacing between the particle patterns and position alters the bending curvature and direction of this composite hydrogel sheet, respectively. Moreover, the orientation (i.e., relative angle) of the particle alignments with respect to the long axis of laser-cut hydrogel strips governs the bending behaviors and the subsequent shape deformation by external stimuli. This acousto-photolithography provides a means of spatiotemporal programming of the internal heterogeneity of composite polymeric systems.

Microscale concert hall acoustics to produce uniform ultrasound stimulation for targeted sonogenetics in hsTRPA1-transfected cells.

The field of ultrasound neuromodulation has rapidly developed over the past decade, a consequence of the discovery of strain-sensitive structures in the membrane and organelles of cells extending into the brain, heart, and other organs. Notably, clinical trials are underway for treating epilepsy using focused ultrasound to elicit an organized local electrical response. A key limitation to this approach is the formation of standing waves within the skull. In standing acoustic waves, the maximum ultrasound intensity spatially varies from near zero to double the mean in one half a wavelength, and has lead to localized tissue damage and disruption of normal brain function while attempting to evoke a broader response. This phenomenon also produces a large spatial variation in the actual ultrasound exposure in tissue, leading to heterogeneous results and challenges with interpreting these effects. One approach to overcome this limitation is presented herein: transducer-mounted diffusers that result in spatiotemporally incoherent ultrasound. Herein, we numerically and experimentally quantified the effect of a diffuser in an enclosed domain, and show that adding the diffuser leads to a two-fold increase in ultrasound responsiveness of hsTRPA1 transfected HEK cells. Furthermore, we demonstrate the diffuser allow us to produce an uniform spatial distribution of pressure in the rodent skull. Collectively, we propose that our approach leads to a means to deliver uniform ultrasound into irregular cavities for sonogenetics.

Nebulization of siRNA for inhalation therapy based on a microfluidic surface acoustic wave platform.

The local delivery of therapeutic small interfering RNA or siRNA to the lungs has the potential to improve the prognosis for patients suffering debilitating lung diseases. Recent advances in materials science have been aimed at addressing delivery challenges including biodistribution, bioavailability and cell internalization, but an equally important challenge to overcome is the development of an inhalation device that can deliver the siRNA effectively to the lung, without degrading the therapeutic itself. Here, we report the nebulization of siRNA, either naked siRNA or complexed with polyethyleneimine (PEI) or a commercial transfection agent, using a miniaturizable acoustomicrofluidic nebulization device. The siRNA solution could be nebulised without significant degradation into an aerosol mist with tunable mean aerodynamic diameters of approximately 3 µm, which is appropriate for deep lung deposition via inhalation. The nebulized siRNA was tested for its stability, as well as its toxicity and gene silencing properties using the mammalian lung carcinoma cell line A549, which demonstrated that the gene silencing capability of siRNA is retained after nebulization. This highlights the potential application of the acoustomicrofluidic device for the delivery of efficacious siRNA via inhalation, either for systemic delivery via the alveolar epithelium or local therapeutic delivery to the lung.

Two pathways are required for ultrasound-evoked behavioral changes in Caenorhabditis elegans.

Ultrasound has been shown to affect the function of both neurons and non-neuronal cells, but, the underlying molecular machinery has been poorly understood. Here, we show that at least two mechanosensitive proteins act together to generate C. elegans behavioral responses to ultrasound stimuli. We first show that these animals generate reversals in response to a single 10 msec pulse from a 2.25 MHz ultrasound transducer. Next, we show that the pore-forming subunit of the mechanosensitive channel TRP-4, and a DEG/ENaC/ASIC ion channel MEC-4, are both required for this ultrasound-evoked reversal response. Further, the trp-4;mec-4 double mutant shows a stronger behavioral deficit compared to either single mutant. Finally, overexpressing TRP-4 in specific chemosensory neurons can rescue the ultrasound-triggered behavioral deficit in the mec-4 null mutant, suggesting that both TRP-4 and MEC-4 act together in affecting behavior. Together, we demonstrate that multiple mechanosensitive proteins likely cooperate to transform ultrasound stimuli into behavioral changes.

Well-free agglomeration and on-demand three-dimensional cell cluster formation using guided surface acoustic waves through a couplant layer.

Three-dimensional cell agglomerates are broadly useful in tissue engineering and drug testing. We report a well-free method to form large (1.4-mm) multicellular clusters using 100-MHz surface acoustic waves (SAW) without direct contact with the media or cells. A fluid couplant is used to transform the SAW into acoustic streaming in the cell-laden media held in a petri dish. The couplant transmits longitudinal sound waves, forming a Lamb wave in the petri dish that, in turn, produces longitudinal sound in the media. Due to recirculation, human embryonic kidney (HEK293) cells in the dish are carried to the center of the coupling location, forming a cluster in less than 10 min. A few minutes later, these clusters may then be translated and merged to form large agglomerations, and even repeatedly folded to produce a roughly spherical shape of over 1.4 mm in diameter for incubation-without damaging the existing intercellular bonds. Calcium ion signaling through these clusters and confocal images of multiprotein junctional complexes suggest a continuous tissue construct: intercellular communication. They may be formed at will, and the method is feasibly useful for formation of numerous agglomerates in a single petri dish.

Robotics and Artificial Intelligence in Endovascular Neurosurgery.

The use of artificial intelligence (AI) and robotics in endovascular neurosurgery promises to transform neurovascular care. We present a review of the recently published neurosurgical literature on artificial intelligence and robotics in endovascular neurosurgery to provide insights into the current advances and applications of this technology. The PubMed database was searched for "neurosurgery" OR "endovascular" OR "interventional" AND "robotics" OR "artificial intelligence" between January 2016 and August 2021. A total of 1296 articles were identified, and after applying the inclusion and exclusion criteria, 38 manuscripts were selected for review and analysis. These manuscripts were divided into four categories: 1) robotics and AI for the diagnosis of cerebrovascular pathology, 2) robotics and AI for the treatment of cerebrovascular pathology, 3) robotics and AI for training in neuroendovascular procedures, and 4) robotics and AI for clinical outcome optimization. The 38 articles presented include 23 articles on AI-based diagnosis of cerebrovascular disease, 10 articles on AI-based treatment of cerebrovascular disease, two articles on AI-based training techniques for neuroendovascular procedures, and three articles reporting AI prediction models of clinical outcomes in vascular disorders of the brain. Innovation with robotics and AI focus on diagnostic efficiency, optimizing treatment and interventional procedures, improving physician procedural performance, and predicting clinical outcomes with the use of artificial intelligence and robotics. Experimental studies with robotic systems have demonstrated safety and efficacy in treating cerebrovascular disorders, and novel microcatheterization techniques may permit access to deeper brain regions. Other studies show that pre-procedural simulations increase overall physician performance. Artificial intelligence also shows superiority over existing statistical tools in predicting clinical outcomes. The recent advances and current usage of robotics and AI in the endovascular neurosurgery field suggest that the collaboration between physicians and machines has a bright future for the improvement of patient care. The aim of this work is to equip the medical readership, in particular the neurosurgical specialty, with tools to better understand and apply findings from research on artificial intelligence and robotics in endovascular neurosurgery.

Sonogenetic control of mammalian cells using exogenous Transient Receptor Potential A1 channels.

Ultrasound has been used to non-invasively manipulate neuronal functions in humans and other animals. However, this approach is limited as it has been challenging to target specific cells within the brain or body. Here, we identify human Transient Receptor Potential A1 (hsTRPA1) as a candidate that confers ultrasound sensitivity to mammalian cells. Ultrasound-evoked gating of hsTRPA1 specifically requires its N-terminal tip region and cholesterol interactions; and target cells with an intact actin cytoskeleton, revealing elements of the sonogenetic mechanism. Next, we use calcium imaging and electrophysiology to show that hsTRPA1 potentiates ultrasound-evoked responses in primary neurons. Furthermore, unilateral expression of hsTRPA1 in mouse layer V motor cortical neurons leads to c-fos expression and contralateral limb responses in response to ultrasound delivered through an intact skull. Collectively, we demonstrate that hsTRPA1-based sonogenetics can effectively manipulate neurons within the intact mammalian brain, a method that could be used across species.

Vacuum exhausted isolation locker (VEIL) to reduce inpatient droplet/aerosol transmission during COVID-19 pandemic.

The vacuum-exhausted isolation locker (VEIL) provides a safety barrier during the care of COVID-19 patients. The VEIL is a 175-L enclosure with exhaust ports to continuously extract air through viral particle filters connected to hospital suction. Our experiments show that the VEIL contains and exhausts exhaled aerosols and droplets.

Ultrasound Mediated Cellular Deflection Results in Cellular Depolarization.

Ultrasound has been used to manipulate cells in both humans and animal models. While intramembrane cavitation and lipid clustering have been suggested as likely mechanisms, they lack experimental evidence. Here, high-speed digital holographic microscopy (kiloHertz order) is used to visualize the cellular membrane dynamics. It is shown that neuronal and fibroblast membranes deflect about 150 nm upon ultrasound stimulation. Next, a biomechanical model that predicts changes in membrane voltage after ultrasound exposure is developed. Finally, the model predictions are validated using whole-cell patch clamp electrophysiology on primary neurons. Collectively, it is shown that ultrasound stimulation directly defects the neuronal membrane leading to a change in membrane voltage and subsequent depolarization. The model is consistent with existing data and provides a mechanism for both ultrasound-evoked neurostimulation and sonogenetic control.

An investigation of maximum particle velocity as a universal invariant-Defined by a statistical measure of failure or plastic energy loss for acoustofluidic applications.

Materials under vibration experience internal stress waves that can cause material failure or energy loss due to inelastic vibration. Traditionally, failure is defined in terms of material acceleration, yet this approach has many drawbacks, principally because it is not invariant with respect to scale, type of vibration, or material choice. Here, the likelihood of failure is instead considered in terms of the maximum vibration or particle velocity for various metals, polymers, and structural materials. The exact relationship between the maximum particle velocity and the maximum induced stress may be derived, but only if one knows the details of the vibration, material, flaws, and geometry. Statistical results with over thousands of individual trials are presented here to demonstrate a wide variety of vibrations across a sufficient variety of these choices. Failure in this context is defined as either fracture or plastic yield, the latter associated with inelastic deformation and energy loss during vibration. If the maximum permissible cyclical stress in material vibration is known, to at least an order of magnitude, the probability of this type of failure may be computed for a range of vibration velocities in each material. The results support the notion that a maximum particle velocity on the order of 1 m/s is a universal and critical limit that, upon exceeding, causes the probability of failure to become significant regardless of the details of the material, geometry, or vibration. We illustrate this in a specific example relevant to acoustofluidics, a simple surface acoustic wave device. The consequences of particle velocity limit analysis can effectively be used in materials and structural engineering to predict when dynamic material particle velocity can cause inelastic losses or failure via brittle fracture, plastic deformation, or fatigue failure.

Soft robotic steerable microcatheter for the endovascular treatment of cerebral disorders.

Catheters used for endovascular navigation in interventional procedures lack dexterity at the distal tip. Neurointerventionists, in particular, encounter challenges in up to 25% of aneurysm cases largely due to the inability to steer and navigate the tip of the microcatheters through tortuous vasculature to access aneurysms. We overcome this problem with submillimeter diameter, hydraulically actuated hyperelastic polymer devices at the distal tip of microcatheters to enable active steerability. Controlled by hand, the devices offer complete 3D orientation of the tip. Using saline as a working fluid, we demonstrate guidewire-free navigation, access, and coil deployment in vivo, offering safety, ease of use, and design flexibility absent in other approaches to endovascular intervention. We demonstrate the ability of our device to navigate through vessels and to deliver embolization coils to the cerebral vessels in a live porcine model. This indicates the potential for microhydraulic soft robotics to solve difficult access and treatment problems in endovascular intervention.