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Electrospray deposition (ESD) uses strong electric fields applied to solutions and dispersions exiting a capillary to produce charged monodisperse droplets driven toward grounded targets. Self-limiting electrospray deposition (SLED) is a phenomenon in which highly directed, uniform, and even 3D coatings can be achieved by trapping charge in the deposited film, redirecting the field lines to uncoated regions of the target. However, when inorganic particles are added to SLED sprays, the buildup of charge required to repel incoming material is disrupted as particle loading increases. Due to its fibril gelling behavior, methylcellulose (MC) SLED can form nanowire morphologies. These wires, when used as a binder, can separate particles and prevent percolation. In this work, a variety of conductive and insulating particles are explored using patterned and un-patterned substrates. This exploration allows us to maximally load particles for high-concentration and highly controlled self-limiting functional sprays. This is demonstrated using Ti 3 C 2 T x MXene to functionalize an interdigitated electrode for use as a supercapacitor., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

Electrospray deposition (ESD) is a promising technique for depositing micro-/nano-scale droplets and particles with high quality and repeatability. It is particularly attractive for surface coating of costly and delicate biomaterials and bioactive compounds. While high efficiency of ESD has only been successfully demonstrated for spraying surfaces larger than the spray plume, this work extends its utility to smaller surfaces. It is shown that by architecting the local "charge landscape", ESD coatings of surfaces smaller than plume size can be achieved. Efficiency approaching 100% is demonstrated with multiple model materials, including biocompatible polymers, proteins, and bioactive small molecules, on both flat and microneedle array targets. UV-visible spectroscopy and high-performance liquid chromatography measurements validate the high efficiency and quality of the sprayed material. Here, we show how this process is an efficient and more competitive alternative to other conformal coating mechanisms, such as dip coating or inkjet printing, for micro-engineered applications., (© 2023. Springer Nature Limited.)

There has been a wealth of research conducted regarding the partitioning of red blood cells (RBCs) at bifurcations within the microvasculature. In previous studies, partitioning has been characterized as either regular partitioning, in which the higher flow rate daughter channel receives a proportionally larger percentage of RBCs, or reverse partitioning, in which the opposite occurs. While there are many examples of network studies in silico , most in vitro work has been conducted using single bifurcation. When microfluidic networks have been used, the channel dimensions are typically greater than 20  μ m, ignoring conditions where RBCs are highly confined. This paper presents a study of RBC partitioning in a network of sequential bifurcations with channel dimensions less than 8  μ m in hydraulic diameter. The study investigated the effect of the volumetric flow rate ratio ( Q* ) at each bifurcation, solution hematocrit, and channel length on the erythrocyte flux ratio ( N* ), a measure of RBC partitioning. We report significant differences in partitioning between upstream and downstream bifurcations even when the flow rate ratio remains the same. Skewness analysis, a measure of cell distribution across the width of a vessel, strongly suggests that immediately following the first bifurcation most RBCs are skewed toward the inner channel wall, leading to preferential RBC perfusion into one daughter channel at the subsequent bifurcation even at higher downstream flow rate ratios. The skewness of RBC distribution following the first bifurcation can either manifest as enhanced regular partitioning or reverse partitioning at the succeeding branch., (© 2022 Author(s).)

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

Mental disorders have high prevalence, but the efficacy of existing therapeutics is limited, in part, because the pathogenic mechanisms remain enigmatic. Current models of neural circuitry include animal models and post-mortem brain tissue, which have allowed enormous progress in understanding the pathophysiology of mental disorders. However, these models limit the ability to assess the functional alterations in short-range and long-range network connectivity between brain regions that are implicated in many mental disorders, e.g., schizophrenia and autism spectrum disorders. This work addresses these limitations by developing an in vitro model of the human brain that models the in vivo cerebral tract environment. In this study, microfabrication and stem cell differentiation techniques were combined to develop an in vitro cerebral tract model that anchors human induced pluripotent stem cell-derived cerebral organoids (COs) and provides a scaffold to promote the formation of a functional connecting neuronal tract. Two designs of a Cerebral Organoid Connectivity Apparatus (COCA) were fabricated using SU-8 photoresist. The first design contains a series of spikes which anchor the CO to the COCA (spiked design), whereas the second design contains flat supporting structures with open holes in a grid pattern to anchor the organoids (grid design); both designs allow effective media exchange. Morphological and functional analyses reveal the expression of key neuronal markers as well as functional activity and signal propagation along cerebral tracts connecting CO pairs. The reported in vitro models enable the investigation of critical neural circuitry involved in neurodevelopmental processes and has the potential to help devise personalized and targeted therapeutic strategies.

This work reports a suction-based cutaneous delivery method for in vivo DNA transfection. Following intradermal Mantoux injection of plasmid DNA in a rat model, a moderate negative pressure is applied to the injection site, a technique similar to Chinese báguàn and Middle Eastern hijama cupping therapies. Strong GFP expression was demonstrated with pEGFP-N1 plasmids where fluorescence was observed as early as 1 hour after dosing. Modeling indicates a strong correlation between focal strain/stress and expression patterns. The absence of visible and/or histological tissue injury contrasts with current in vivo transfection systems such as electroporation. Specific utility was demonstrated with a synthetic SARS-CoV-2 DNA vaccine, which generated host humoral immune response in rats with notable antibody production. This method enables an easy-to-use, cost-effective, and highly scalable platform for both laboratorial transfection needs and clinical applications for nucleic acid–based therapeutics and vaccines.

There is a long history of research examining red blood cell (RBC) partitioning in microvasculature bifurcations. These studies commonly report results describing partitioning that exists as either regular partitioning, which occurs when the RBC flux ratio is greater than the bulk fluid flowrate ratio, or reverse partitioning when the RBC flux ratio is less than or equal to that of the bulk fluid flowrate. This paper presents a study of RBC partitioning in a single bifurcating microchannel with dimensions of 6 to 16 μm, investigating the effects of hematocrit, channel width, daughter channel flowrate ratio, and bifurcation angle. The erythrocyte flux ratio, N*, manifests itself as either regular or reverse partitioning, and time-dependent partitioning is much more dynamic, occurring as both regular and reverse partitioning. We report a significant reduction in the well-known sigmoidal variation of the erythrocyte flux ratio (N*) versus the volumetric flowrate ratio (Q*), partitioning behavior with increasing hematocrit in microchannels when the channel dimensions are comparable with cell size. RBCs "lingering" or jamming at the bifurcation were also observed and quantified in vitro. Results from trajectory analyses suggest that the RBC position in the feeder channel strongly affects both partitioning and lingering frequency of RBCs, with both being significantly reduced when RBCs flow on streamlines near the edge of the channel as opposed to the center of the channel. Furthermore, our experiments suggest that even at low Reynolds number, partitioning is affected by the bifurcation angle by increasing cell-cell interactions. The presented results provide further insight into RBC partitioning as well as perfusion throughout the microvasculature., (© 2021 International Center for Artificial Organs and Transplantation and Wiley Periodicals LLC.)

The mechanical behavior and cortical tension of single cells are analyzed using electrodeformation relaxation. Four types of cells, namely, MCF-10A, MCF-7, MDA-MB-231, and GBM, are studied, with pulse durations ranging from 0.01 to 10 s. Mechanical response in the long-pulse regime is characterized by a power-law behavior, consistent with soft glassy rheology resulting from unbinding events within the cortex network. In the subsecond short-pulse regime, a single timescale well describes the process and indicates the naive tensioned (prestressed) state of the cortex with minimal force-induced alteration. A mathematical model is employed and the simple ellipsoidal geometry allows for use of an analytical solution to extract the cortical tension. At the shortest pulse of 0.01 s, tensions for all four cell types are on the order of 10^{-2} N/m.

Intracortical neural probes are a key enabling technology for acquiring high fidelity neural signals within the cortex. They are viewed as a crucial component of brain-computer interfaces (BCIs) in order to record electrical activities from neurons within the brain. Smaller, more flexible, polymer-based probes have been investigated for their potential to limit the acute and chronic neural tissue response. Conventional methods of patterning electrodes and connecting traces on a single supporting layer can limit the number of recording sites which can be defined, particularly when designing narrower probes. We present a novel strategy of increasing the number of recording sites without proportionally increasing the size of the probe by using a multilayer fabrication process to vertically layer recording traces on multiple Parylene support layers, allowing more recording traces to be defined on a smaller probe width. Using this approach, we are able to define 16 electrodes on 4 supporting layers (4 electrodes per layer), each with a 30 μ m diameter recording window and 5 μ m wide connecting trace defined by conventional LWUV lithography, on an 80 μ m wide by 9 μ m thick microprobe. Prior to in vitro and in vivo validation, the multilayer probes are electrically characterized via impedance spectroscopy and evaluating crosstalk between adjacent layers. Demonstration of acute in vitro recordings in a cerebral organoid model and in vivo recordings in a murine model indicate the probe's capability for single unit recordings. This work demonstrates the ability to fabricate smaller, more compliant neural probes without sacrificing electrode density.

Electrospray deposition (ESD) applies a high voltage to liquids flowing through narrow capillaries to produce monodisperse generations of droplets down to hundreds of nanometers in diameter, each carrying a small amount of the delivered solute. This deposition method has been combined with insulated stencil masks for fabricating micropatterns by spraying solutions containing nanoparticles, polymers, or biomaterials. To optimize the fabrication process for micro-coatings, a self-limiting electrospray deposition (SLED) method has recently been developed. Here, we combine SLED with a pre-existing patterned polymer film to study SLED's fundamental behavior in a bilayer geometry. SLED has been observed when glassy insulating materials are sprayed onto conductive substrates, where a thickness-limited film forms as charge accumulates and repels the arrival of additional charged droplets. In this study, polystyrene (PS), Parylene C, and SU-8 thin films of varying thickness on silicon are utilized as insulated spraying substrates. Polyvinylpyrrolidone (PVP), a thermoplastic polymer is sprayed below its glass transition temperature (T g ) to investigate the SLED behavior on the pre-deposited insulating films. Furthermore, to examine the effects of in-plane confinement on the spray, a microhole array patterned onto the PS thin film by laser dewetting was sprayed with dyed PVP in the SLED mode. This was then extended to an unmasked electrode array showing that masked SLED and laser dewetting could be used to target microscale regions of conventionally-patterned electronics.

Proper brain function relies on the precise arrangement and flow of information between diverse neural subtypes. Developing improved human cell-based models which faithfully mimic biologically relevant connectivity patterns may improve drug screening efforts given the limited success of animal models to predict safety and efficacy of therapeutics in human clinical trials. To address this need, we have developed experimental models of defined neural circuitries through the compartmentalization of neuronal cell subtypes in a 96 well plate-based platform where each microwell is divided into two compartments connected by microchannels allowing high-throughput screening (HTS) of small molecules. We demonstrate that we can generate subtype-specific excitatory and inhibitory induced neuronal cells (iNs) from human stem cell lines and that these neurons form robust functional circuits with defined connectivity. Through the use of the genetically encoded calcium indicator GCaMP6f, we monitor calcium ion transients generated during neuronal firing between and within compartments. We further demonstrate functionality of the circuit by perturbing network activity through the addition of glutamate receptor blockers using automated liquid handling. Lastly, we show that we can stimulate network activity in defined neuronal subtypes through the expression of the designer receptor exclusively activated by designer drugs (DREADD) hM3Dq and application of the ligand clozapine-N-oxide (CNO). Our results demonstrate the formation of functional neural circuits in a high-throughput platform that is compatible with compound screening, representing an important step towards developing new screening platforms for studying and ultimately treating psychiatric brain disorders that arise from disordered neural circuit function.

Electroporation is an electro-physical, non-viral approach to perform DNA, RNA, and protein transfections of cells. Upon application of an electric field, the cell membrane is compromised, allowing the delivery of exogenous materials into cells. Cell viability and electro-transfection efficiency (eTE) are dependent on various experimental factors, including pulse waveform, vector concentration, cell type/density, and electroporation buffer properties. In this work, the effects of buffer composition on cell viability and eTE were systematically explored for plasmid DNA encoding green fluorescent protein following electroporation of 3T3 fibroblasts. A HEPES-based buffer was used in conjunction with various salts and sugars to modulate conductivity and osmolality, respectively. Pulse applications were chosen to maintain constant applied electrical energy (J) or total charge flux (C/m 2 ). The energy of the pulse application primarily dictated cell viability, with Mg 2+ -based buffers expanding the reversible electroporation range. The enhancement of viability with Mg 2+ -based buffers led to the hypothesis that this enhancement is due to ATPase activation via re-establishing ionic homeostasis. We show preliminary evidence for this mechanism by demonstrating that the enhanced viability is eliminated by introducing lidocaine, an ATPase inhibitor. However, Mg 2+ also hinders eTE compared to K + -based buffers. Collectively, the results demonstrate that the rational selection of pulsing conditions and buffer compositions are critical for the design of electroporation protocols to maximize viability and eTE.

Mild traumatic brain injury (mTBI) is a frequently overlooked public health concern that is difficult to diagnose and treat. Diffuse axonal injury (DAI) is a common mTBI neuropathology in which axonal shearing and stretching induces breakdown of the cytoskeleton, impaired axonal trafficking, axonal degeneration, and cognitive dysfunction. DAI is becoming recognized as a principal neuropathology of mTBI with supporting evidence from animal model, human pathology, and neuroimaging studies. As mitochondrial dysfunction and calcium overload are critical steps in secondary brain and axonal injury, we investigated changes in protein expression of potential targets following mTBI using an in vivo controlled cortical impact model. We show upregulated expression of sodium calcium exchanger1 (NCX1) in the hippocampus and cortex at distinct time points post-mTBI. Expression of dynamin-related protein1 (Drp1), a GTPase responsible for regulation of mitochondrial fission, also changes differently post-injury in the hippocampus and cortex. Using an in vitro model of DAI previously reported by our group, we tested whether pharmacological inhibition of NCX1 by SN-6 and of dynamin1, dynamin2, and Drp1 by dynasore mitigates secondary damage. Dynasore and SN-6 attenuate stretch injury-induced swelling of axonal varicosities and mitochondrial fragmentation. In addition, we show that dynasore, but not SN-6, protects against H 2 O 2 -induced damage in an organotypic oxidative stress model. As there is currently no standard treatment to mitigate cell damage induced by mTBI and DAI, this work highlights two potential therapeutic targets for treatment of DAI in multiple models of mTBI and DAI.

Neuropsychiatric disorders have traditionally been difficult to study due to the complexity of the human brain and limited availability of human tissue. Induced pluripotent stem (iPS) cells provide a promising avenue to further our understanding of human disease mechanisms, but traditional 2D cell cultures can only provide a limited view of the neural circuits. To better model complex brain neurocircuitry, compartmentalized culturing systems and 3D organoids have been developed. Early compartmentalized devices demonstrated how neuronal cell bodies can be isolated both physically and chemically from neurites. Soft lithographic approaches have advanced this approach and offer the tools to construct novel model platforms, enabling circuit-level studies of disease, which can accelerate mechanistic studies and drug candidate screening. In this review, we describe some of the common technologies used to develop such systems and discuss how these lithographic techniques have been used to advance our understanding of neuropsychiatric disease. Finally, we address other in vitro model platforms such as 3D culture systems and organoids and compare these models with compartmentalized models. We ask important questions regarding how we can further harness iPS cells in these engineered culture systems for the development of improved in vitro models. Developmental Dynamics 248:65-77, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Multicellular aggregates are an excellent model system to explore the role of tissue biomechanics in specifying multicellular reorganization during embryonic developments and malignant invasion. Tissue-like spheroids, when subjected to a compressive force, are known to exhibit liquid-like behaviors at long timescales (hours), largely because of cell rearrangements that serve to effectively dissipate the applied stress. At short timescales (seconds to minutes), before cell rearrangement, the mechanical behavior is strikingly different. The current work uses shape relaxation to investigate the structural characteristics of aggregates and discovers two coherent timescales: one on the order of seconds, the other tens of seconds. These timescales are universal, conserved across a variety of tested species, and persist despite great differences in other properties such as tissue surface tension and adhesion. A precise mathematical theory is used to correlate the timescales with mechanical properties and reveals that aggregates have a relatively strong envelope and an unusually "soft" interior (weak bulk elastic modulus). This characteristic is peculiar, considering that both layers consist of identical units (cells), but is consistent with the fact that this structure can engender both structural integrity and the flexibility required for remodeling. In addition, tissue surface tension, elastic modulus, and viscosity are proportional to each other. Considering that these tissue-level properties intrinsically derive from cellular-level properties, the proportionalities imply precise coregulation of the latter and in particular of the tension on the cell-medium and cell-cell interfaces., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Objective: Despite the feasibility of short-term neural recordings using implantable microelectrodes, attaining reliable, chronic recordings remains a challenge. Most neural recording devices suffer from a long-term tissue response, including gliosis, at the device-tissue interface. It was hypothesized that smaller, more flexible intracortical probes would limit gliosis by providing a better mechanical match with surrounding tissue., Approach: This paper describes the in vivo evaluation of flexible parylene microprobes designed to improve the interface with the adjacent neural tissue to limit gliosis and thereby allow for improved recording longevity. The probes were coated with an ultrafast degrading tyrosine-derived polycarbonate (E5005(2K)) polymer that provides temporary mechanical support for device implantation, yet degrades within 2 h post-implantation. A parametric study of probes of varying dimensions and polymer coating thicknesses were implanted in rat brains. The glial tissue response and neuronal loss were assessed from 72 h to 24 weeks post-implantation via immunohistochemistry., Main Results: Experimental results suggest that both probe and polymer coating sizes affect the extent of gliosis. When an appropriate sized coating dimension (100 µm  ×  100 µm) and small probe (30 µm  ×  5 µm) was implanted, a minimal post-implantation glial response was observed. No discernible gliosis was detected when compared to tissue where a sham control consisting of a solid degradable polymer shuttle of the same dimensions was inserted. A larger polymer coating (200 µm  ×  200 µm) device induced a more severe glial response at later time points, suggesting that the initial insertion trauma can affect gliosis even when the polymer shuttle degrades rapidly. A larger degree of gliosis was also observed when comparing a larger sized probe (80 µm  ×  5 µm) to a smaller probe (30 µm  ×  5 µm) using the same polymer coating size (100 µm  ×  100 µm). There was no significant neuronal loss around the implantation sites for most device candidates except the group with largest polymer coating and probe sizes., Significance: These results suggest that: (1) the degree of mechanical trauma at device implantation and mechanical mismatches at the probe-tissue interface affect long term gliosis; (2) smaller, more flexible probes may minimize the glial response to provide improved tissue biocompatibility when used for chronic neural signal recording; and (3) some degree of glial scarring did not significantly affect neuronal distribution around the probe.

Traumatic brain injury (TBI) affects 5.3 million people in the United States, and there are 12,500 new cases of spinal cord injury (SCI) every year. There is yet a significant need for in vitro models of TBI and SCI in order to understand the biological mechanisms underlying central nervous system (CNS) injury and to identify and test therapeutics to aid in recovery from neuronal injuries. While TBI or SCI studies have been aided with traditional in vivo and in vitro models, the innate limitations in specificity of injury, isolation of neuronal regions, and reproducibility of these models can decrease their usefulness in examining the neurobiology of injury. Microfluidic devices provide several advantages over traditional methods by allowing researchers to (1) examine the effect of injury on specific neural components, (2) fluidically isolate neuronal regions to examine specific effects on subcellular components, and (3) reproducibly create a variety of injuries to model TBI and SCI. These microfluidic devices are adaptable for modeling a wide range of injuries, and in this review, we will examine different methodologies and models recently utilized to examine neuronal injury. Specifically, we will examine vacuum-assisted axotomy, physical injury, chemical injury, and laser-based axotomy. Finally, we will discuss the benefits and downsides to each type of injury model and discuss how researchers can use these parameters to pick a particular microfluidic device to model CNS injury., (© 2017 Wiley Periodicals, Inc.)

Flow cytometry is an invaluable tool utilized in modern biomedical research and clinical applications requiring high throughput, high resolution particle analysis for cytometric characterization and/or sorting of cells and particles as well as for analyzing results from immunocytometric assays. In recent years, research has focused on developing microfluidic flow cytometers with the motivation of creating smaller, less expensive, simpler, and more autonomous alternatives to conventional flow cytometers. These devices could ideally be highly portable, easy to operate without extensive user training, and utilized for research purposes and/or point-of-care diagnostics especially in limited resource facilities or locations requiring on-site analyses. However, designing a device that fulfills the criteria of high throughput analysis, automation and portability, while not sacrificing performance is not a trivial matter. This review intends to present the current state of the field and provide considerations for further improvement by focusing on the key design components of microfluidic flow cytometers. The recent innovations in particle focusing and detection strategies are detailed and compared. This review outlines performance matrix parameters of flow cytometers that are interdependent with each other, suggesting trade offs in selection based on the requirements of the applications. The ongoing contribution of microfluidics demonstrates that it is a viable technology to advance the current state of flow cytometry and develop automated, easy to operate and cost-effective flow cytometers., Competing Interests: CONFLICTS OF INTEREST There are no conflicts of interest to declare.

The construction of biomaterials with which to limit the growth of cells or to limit the adsorption of proteins is essential for understanding biological phenomena. Here, we describe a novel method to simply and easily create thin layers of poly (2-hydroxyethyl methacrylate) (p-HEMA) for protein and cellular patterning via etching with ethanol and microfluidic devices. First, a cell culture surface or glass coverslip is coated with p-HEMA. Next, a polydimethylsiloxane (PDMS) microfluidic is placed onto the p-HEMA surface, and ethanol is aspirated through the device. The PDMS device is removed, and the p-HEMA surface is ready for protein adsorption or cell plating. This method allows for the fabrication of 0.3 µm thin layers of p-HEMA, which can be etched to 10 µm wide channels. Furthermore, it creates regions of differential protein adhesion, as shown by Coomassie staining and fluorescent labeling, and cell adhesion, as demonstrated by C2C12 myoblast growth. This method is simple, versatile, and allows biologists and bioengineers to manipulate regions for cell culture adhesion and growth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:243-248, 2018., (© 2017 American Institute of Chemical Engineers.)

Synapse formation analyses can be performed by imaging and quantifying fluorescent signals of synaptic markers. Traditionally, these analyses are done using simple or multiple thresholding and segmentation approaches or by labor-intensive manual analysis by a human observer. Here, we describe Intellicount , a high-throughput, fully-automated synapse quantification program which applies a novel machine learning (ML)-based image processing algorithm to systematically improve region of interest (ROI) identification over simple thresholding techniques. Through processing large datasets from both human and mouse neurons, we demonstrate that this approach allows image processing to proceed independently of carefully set thresholds, thus reducing the need for human intervention. As a result, this method can efficiently and accurately process large image datasets with minimal interaction by the experimenter, making it less prone to bias and less liable to human error. Furthermore, Intellicount is integrated into an intuitive graphical user interface (GUI) that provides a set of valuable features, including automated and multifunctional figure generation, routine statistical analyses, and the ability to run full datasets through nested folders, greatly expediting the data analysis process.

Neurocircuits in the human brain govern complex behavior and involve connections from many different neuronal subtypes from different brain regions. Recent advances in stem cell biology have enabled the derivation of patient-specific human neuronal cells of various subtypes for the study of neuronal function and disease pathology. Nevertheless, one persistent challenge using these human-derived neurons is the ability to reconstruct models of human brain circuitry. To overcome this obstacle, we have developed a compartmentalized microfluidic device, which allows for spatial separation of cell bodies of different human-derived neuronal subtypes (excitatory, inhibitory and dopaminergic) but is permissive to the spreading of projecting processes. Induced neurons (iNs) cultured in the device expressed pan-neuronal markers and subtype specific markers. Morphologically, we demonstrate defined synaptic contacts between selected neuronal subtypes by synapsin staining. Functionally, we show that excitatory neuronal stimulation evoked excitatory postsynaptic current responses in the neurons cultured in a separate chamber.

Background: Dispersal of glioblastoma (GBM) cells leads to recurrence and poor prognosis. Accordingly, molecular pathways involved in dispersal are potential therapeutic targets. The mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) pathway is commonly dysregulated in GBM, and targeting this pathway with MEK inhibitors has proven effective in controlling tumor growth. Since this pathway also regulates ECM remodeling and actin organization - processes crucial to cell adhesion, substrate attachment, and cell motility - the aim of this study was to determine whether inhibiting this pathway could also impede dispersal., Methods: A variety of methods were used to quantify the effects of the MEK inhibitor, PD0325901, on potential regulators of dispersal. Cohesion, stiffness and viscosity were quantified using a method based on ellipsoid relaxation after removal of a deforming external force. Attachment strength, cell motility, spheroid dispersal velocity, and 3D growth rate were quantified using previously described methods., Results: We show that PD0325901 significantly increases aggregate cohesion, stiffness, and viscosity but only when tumor cells have access to high concentrations of fibronectin. Treatment also results in reorganization of actin from cortical into stress fibers, in both 2D and 3D culture. Moreover, drug treatment localized pFAK at sites of cell-substratum adhesion. Collectively, these changes resulted in increased strength of substrate attachment and decreased motility, a decrease in aggregate dispersal velocity, and in a marked decrease in growth rate of both 2D and 3D cultures., Conclusions: Inhibition of the MAPK/ERK pathway by PD0325901 may be an effective therapy for reducing dispersal and growth of GBM cells.

Single-unit recording neural probes have significant advantages towards improving signal-to-noise ratio and specificity for signal acquisition in brain-to-computer interface devices. Long-term effectiveness is unfortunately limited by the chronic injury response, which has been linked to the mechanical mismatch between rigid probes and compliant brain tissue. Small, flexible microelectrodes may overcome this limitation, but insertion of these probes without buckling requires supporting elements such as a stiff coating with a biodegradable polymer. For these coated probes, there is a design trade-off between the potential for successful insertion into brain tissue and the degree of trauma generated by the insertion. The objective of this study was to develop and validate a finite element model (FEM) to simulate insertion of coated neural probes of varying dimensions and material properties into brain tissue. Simulations were performed to predict the buckling and insertion forces during insertion of coated probes into a tissue phantom with material properties of brain. The simulations were validated with parallel experimental studies where probes were inserted into agarose tissue phantom, ex vivo chick embryonic brain tissue, and ex vivo rat brain tissue. Experiments were performed with uncoated copper wire and both uncoated and coated SU-8 photoresist and Parylene C probes. Model predictions were found to strongly agree with experimental results (<10% error). The ratio of the predicted buckling force-to-predicted insertion force, where a value greater than one would ideally be expected to result in successful insertion, was plotted against the actual success rate from experiments. A sigmoidal relationship was observed, with a ratio of 1.35 corresponding to equal probability of insertion and failure, and a ratio of 3.5 corresponding to a 100% success rate. This ratio was dubbed the "safety factor", as it indicated the degree to which the coating should be over-designed to ensure successful insertion. Probability color maps were generated to visually compare the influence of design parameters. Statistical metrics derived from the color maps and multi-variable regression analysis confirmed that coating thickness and probe length were the most important features in influencing insertion potential. The model also revealed the effects of manufacturing flaws on insertion potential.

Immunoassays are widely utilized due to their ability to quantify a vast assortment of biomolecules relevant to biological research and clinical diagnostics. Recently, immunoassay capabilities have been improved by the development of multiplex assays that simultaneously measure multiple analytes in a single sample. However, these assays are hindered by high costs of reagents and relatively large sample requirements. For example, in vitro screening systems currently dedicate individual wells to each time point of interest and this limitation is amplified in screening studies when the investigation of many experimental conditions is necessary; resulting in large volumes for analysis, a correspondingly high cost and a limited temporal experimental design. Microfluidics based immunoassays have been developed in order to overcome these drawbacks. Together, previous studies have demonstrated on-chip assays with either a large dynamic range, high performance sensitivity, and/or the ability to process samples in parallel on a single chip. In this report, we develop a multiplex immunoassay possessing all of these parallel characteristics using commercially available reagents, which allows the analytes of interest to be easily changed. The device presented can measure 6 proteins in 32 samples simultaneously using only 4.2 μL of sample volume. High quality standard curves are generated for all 6 analytes included in the analysis, and spiked samples are quantified throughout the working range of the assay. In addition, we demonstrate a strong correlation (R(2) = 0.8999) between in vitro supernatant measurements using our device and those obtained from a bench-top multiplex immunoassay. Finally, we describe cytokine secretion in an in vitro inflammatory hippocampus culture system, establishing proof-of-concept of the ability to use this platform as an in vitro screening tool. The low-volume, multiplexing abilities of the microdevice described in this report could be broadly applied to numerous situations where sample volumes and costs are limiting.

Electroporation is of interest for many drug-delivery and gene-therapy applications. Prior studies have shown that a two-pulse-electroporation protocol consisting of a short-duration, high-voltage first pulse followed by a longer, low-voltage second pulse can increase delivery efficiency and preserve viability. In this work the effects of the field strength of the first and second pulses and the inter-pulse delay time on the delivery of two different-sized Fluorescein-Dextran (FD) conjugates are investigated. A series of two-pulse-electroporation experiments were performed on 3T3-mouse fibroblast cells, with an alternating-current first pulse to permeabilize the cell, followed by a direct-current second pulse. The protocols were rationally designed to best separate the mechanisms of permeabilization and electrophoretic transport. The results showed that the delivery of FD varied strongly with the strength of the first pulse and the size of the target molecule. The delivered FD concentration also decreased linearly with the logarithm of the inter-pulse delay. The data indicate that membrane resealing after electropermeabilization occurs rapidly, but that a non-negligible fraction of the pores can be reopened by the second pulse for delay times on the order of hundreds of seconds. The role of the second pulse is hypothesized to be more than just electrophoresis, with a minimum threshold field strength required to reopen nano-sized pores or defects remaining from the first pulse. These results suggest that membrane electroporation, sealing, and re-poration is a complex process that has both short-term and long-term components, which may in part explain the wide variation in membrane-resealing times reported in the literature., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We report a fabrication process for coating neural probes with an ultrafast degrading polymer to create consistent and reproducible devices for neural tissue insertion. The rigid polymer coating acts as a probe insertion aid, but resorbs within hours post-implantation. Despite the feasibility for short term neural recordings from currently available neural prosthetic devices, most of these devices suffer from long term gliosis, which isolates the probes from adjacent neurons, increasing the recording impedance and stimulation threshold. The size and stiffness of implanted probes have been identified as critical factors that lead to this long term gliosis. Smaller, more flexible probes that match the mechanical properties of brain tissue could allow better long term integration by limiting the mechanical disruption of the surrounding tissue during and after probe insertion, while being flexible enough to deform with the tissue during brain movement. However, these small flexible probes inherently lack the mechanical strength to penetrate the brain on their own. In this work, we have developed a micromolding method for coating a non-functional miniaturized SU-8 probe with an ultrafast degrading tyrosine-derived polycarbonate (E5005(2K)). Coated, non-functionalized probes of varying dimensions were reproducibly fabricated with high yields. The polymer erosion/degradation profiles of the probes were characterized in vitro. The probes were also mechanically characterized in ex vivo brain tissue models by measuring buckling and insertion forces during probe insertion. The results demonstrate the ability to produce polymer coated probes of consistent quality for future in vivo use, for example to study the effects of different design parameters that may affect tissue response during long term chronic intra-cortical microelectrode neural recordings.

Immunoassays are one of the most versatile and widely performed biochemical assays and, given their selectivity and specificity, are used in both clinical and research settings. However, the high cost of reagents and relatively large sample volumes constrain the integration of immunoassays into many applications. Scaling the assay down within microfluidic devices can alleviate issues associated with reagent and sample consumption. However, in many cases a new device is designed and empirically optimized for each specific analyte, a costly and time consuming approach. In this paper, we report the development of a microfluidic bead-based immunoassay which, using antibody coated microbeads, can potentially detect any analyte or combination of analytes for which antibody coated microbeads can be generated. We also developed a computational reaction model and optimization algorithm that can be used to optimize the device for any analyte. We applied this technique to develop a low volume IL-6 immunoassay with high sensitivity (358 fM, 10 pg/mL) and a large dynamic range (4 orders of magnitude). This device design and optimization technique can be used to design assays for any protein with an available antibody and can be used with a large number of applications including biomarker discovery, temporal in vitro studies using a reduced number of cells and reagents, and analysis of scarce biological samples in animal studies and clinical research settings.

The efficacy of electroporation is known to vary significantly across a wide variety of biological research and clinical applications, but as of this writing, a generalized approach to simultaneously improve efficiency and maintain viability has not been available in the literature. To address that discrepancy, we here outline an approach that is based on the mapping of the scaling relationships among electroporation-mediated molecular delivery, cellular viability, and electric pulse parameters. The delivery of Fluorescein-Dextran into 3T3 mouse fibroblast cells was used as a model system. The pulse was rationally split into two sequential phases: a first precursor for permeabilization, followed by a second one for molecular delivery. Extensive data in the parameter space of the second pulse strength and duration were collected and analyzed with flow cytometry. The fluorescence intensity correlated linearly with the second pulse duration, confirming the dominant role of electrophoresis in delivery. The delivery efficiency exhibited a characteristic sigmoidal dependence on the field strength. An examination of short-term cell death using 7-Aminoactinomycin D demonstrated a convincing linear correlation with respect to the electrical energy. Based on these scaling relationships, an optimal field strength becomes identifiable. A model study was also performed, and the results were compared with the experimental data to elucidate underlying mechanisms. The comparison reveals the existence of a critical transmembrane potential above which delivery with the second pulse becomes effective. Together, these efforts establish a general route to enhance the functionality of electroporation., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A transient analysis to quantify droplet deformation under DC electric fields is presented. The full Taylor-Melcher leaky dielectric model is employed where the charge relaxation time is considered to be finite. The droplet is assumed to be spheroidal in shape for all times. The main result is an ODE governing the evolution of the droplet aspect ratio. The model is validated by extensively comparing predicted deformation with both previous theoretical and numerical studies, and with experimental data. Furthermore, the effects of parameters and stresses on deformation characteristics are systematically analyzed taking advantage of the explicit formulas on their contributions. The theoretical framework can be extended to study similar problems, e.g., vesicle electrodeformation and relaxation.

Myoblast fusion into functionally distinct myotubes, and their subsequent integration with the nervous system, is a poorly understood phenomenon with important applications in basic science research, skeletal muscle tissue engineering, and cell-based biosensor development. We have previously demonstrated the ability of microelectrode arrays (MEAs) to record the extracellular action potentials of myotubes, and we have shown that this information reveals the presence of multiple, electrophysiologically independent myotubes even in unstructured cultures where there is extensive physical contact between cells (Langhammer et al., Biotechnol Prog 27:891-895, 2011). In this paper, we explore the ability of microscale topographical trenches to guide the myoblast alignment and fusion processes and use our findings to create a substrate-embedded MEA containing topographical trenches that are able to direct myotube contractility to specific locations. By combining substrate-embedded MEA technology with topographical patterns, we have developed a lab-on-a-chip test bed for the non-invasive examination of myotubes.

This work demonstrates the use of a continuous online monitoring system for tracking systemic inflammation biomarkers during cardiopulmonary bypass (CPB) procedures. The ability to monitor inflammation biomarkers during CPB will allow surgical teams to actively treat inflammation and reduce harmful effects on postoperative morbidity and mortality, enabling improved patient outcomes. A microfluidic device has been designed which allows automation of the individual processing steps of a microbead immunoassay to allow continuous tracking of antigen concentrations. Preliminary experiments have demonstrated that the results produced by the microimmunoassay are comparable to results produced from a standard enzyme-linked immunosorbent assay (r = 0.98). Additionally, integration of the assay with a simulated CPB circuit has been demonstrated with temporal tracking of C3a concentrations within blood continuously sampled from the circuit. The presented work describes the motivation, design challenges, and preliminary experimental results of this project., (© 2013, Copyright the Authors. Artificial Organs © 2013, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

A microfluidic platform is presented which fully automates all incubation steps of a three-stage, multiplexed magnetic bead immunoassay, such as the Luminex ® xMAP technology. Magnetic actuation is used to transfer the microbeads between co-infused adjacent laminar flow streams to transport the beads into and out of incubation and wash solutions, with extended incubation channels to allow sufficient bead incubation times (1-30 min, commonly 5 min per stage) to enable high-sensitivity. The serial incubation steps of the immunoassay are completed in succession within the device with no operator interaction, and the continuous flow operation with magnetic bead transfer defines the incubation sequencing requiring no external fluidic controls beyond syringe pump infusion. The binding kinetics of the assay is empirically characterized to determine the required incubation times for specific assay sensitivities in the range 1 pg/ml to 100 ng/ml. By using a Luminex ® xMAP duplex assay, concurrent detection of IL-6 and TNF-α was demonstrated on-chip with a detection range 10 pg/ml to 1 ng/ml. This technology enables rapid automation of magnetic microbead assays, and has the potential to perform continuous concentration monitoring.

Sample pre-concentration can be a critical element to improve sensitivity of integrated microchip assays. In this work a converging Y-inlet microfluidic channel with integrated coplanar electrodes was used to investigate transverse DNA and protein migration under uniform direct current (DC) electric fields to assess the ability to concentrate a sample prior to other enzymatic modifications or capillary electrophoretic separations. Employing a pressure-driven flow to perfuse the microchannel, negatively charged samples diluted in low and high ionic strength buffers were co-infused with a receiving buffer of the same ionic strength into a main daughter channel. Experimental results demonstrated that, depending of the buffer selection, different DNA migration and accumulation dynamics were seen. Charged analytes could traverse the channel width and accumulate at the positive bias electrode in a low electroosmotic mobility, high electrophoretic mobility, high ionic strength buffer or migrated towards an equilibrium position within the channel in a high electroosmotic mobility, high electrophoretic mobility, low ionic strength buffer. The various migration behaviours are the result of a balance between the electrophoretic force and a drag force induced by a recirculating electroosmotic flow generated across the channel width due to the bounding walls. Under continuous flow conditions, DNA samples were concentrated several-fold by balancing these transverse electrokinetic forces. The electrokinetic trapping technique presented here is a simple technique which could be expanded to concentrate or separate other analytes as a preconditioning step for downstream processes.

Cell-laden microfluidic devices have broad potential in various biomedical applications, including tissue engineering and drug discovery. However, multiple difficulties encountered while culturing cells within devices affecting cell viability, proliferation, and behavior has complicated their use. While active perfusion systems have been used to overcome the diffusive limitations associated with nutrient delivery into microchannels to support longer culture times, these systems can result in non-uniform oxygen and nutrient delivery and subject cells to shear stresses, which can affect cell behavior. Additionally, histological analysis of cell cultures within devices is generally laborious and yields inconsistent results due to difficulties in delivering labeling agents in microchannels. Herein, we describe a simple, cost-effective approach to preserve cell viability and simplify labeling within microfluidic networks without the need for active perfusion. Instead of bonding a microfluidic network to glass, PDMS, or other solid substrate, the network is bonded to a semi-permeable nanoporous membrane. The membrane-sealed devices allow free exchange of proteins, nutrients, buffers, and labeling reagents between the microfluidic channels and culture media in static culture plates under sterile conditions. The use of the semi-permeable membrane dramatically simplifies microniche cell culturing while avoiding many of the complications which arise from perfusion systems.

This report describes the design, fabrication, and testing of a cross-flow filtration microdevice, for the continuous extraction of blood plasma from a circulating whole blood sample in a clinically relevant environment to assist in continuous monitoring of a patient's inflammatory response during cardiac surgeries involving cardiopulmonary bypass (CPB) procedures (about 400,000 adult and 20,000 pediatric patients in the United States per year). The microfiltration system consists of a two-compartment mass exchanger with two aligned sets of PDMS microchannels, separated by a porous polycarbonate (PCTE) membrane. Using this microdevice, blood plasma has been continuously separated from blood cells in a real-time manner with no evidence of bio-fouling or cell lysis. The technology is designed to continuously extract plasma containing diagnostic plasma proteins such as complements and cytokines using a significantly smaller blood volume as compared to traditional blood collection techniques. The microfiltration device has been tested using a simulated CPB circulation loop primed with donor human blood, in a manner identical to a clinical surgical setup, to collect plasma fractions in order to study the effects of CPB system components and circulation on immune activation during extracorporeal circulatory support. The microdevice, with 200 nm membrane pore size, was connected to a simulated CPB circuit, and was able to continuously extract ~15% pure plasma volume (100% cell-free) with high sampling frequencies which could be analyzed directly following collection with no need to further centrifuge or modify the fraction. Less than 2.5 ml total plasma volume was collected over a 4 h sampling period (less than one Vacutainer blood collection tube volume). The results tracked cytokine concentrations collected from both the reservoir and filtrate samples which were comparable to those from direct blood draws, indicating very high protein recovery of the microdevice. Additionally, the cytokine concentration increased significantly compared to baseline values over the circulation time for all cytokines analyzed. The high plasma protein recovery (over 80%), no indication of hemolysis and low level of biofouling on the membrane surface during the experimental period (over 4 h) were all indications of effective and reliable device performance for future clinical applications. The simple and robust design and operation of these devices allow operation over a wide range of experimental flow conditions and blood hematocrit levels to allow surgeons and clinicians autonomous usage in a clinical environment to better understand the mechanisms of injury resulting from cardiac surgery, and allow early interventions in patients with excessive postoperative complications to improve surgical outcomes. Ultimately, monolithic integration of this microfiltration device with a continuous microimmunoassay would create an integrated microanalysis system for tracking inflammation biomarkers concentrations in patients for point-of-care diagnostics, reducing blood analysis times, costs and volume of blood samples required for repeated assays.

Insulin pens are used by approximately half of worldwide insulin users. Insulin pens have made insulin injections easier compared to traditional vial and syringe injections. In an article in this issue of Journal of Diabetes Science and Technology, Dr. Asakura discusses several important design parameters, which are considered during refillable insulin-injection pen design. Ease of cartridge replacement, insulin-dose setting dial use, injection, and prominence of audible clicks can all affect overall dose accuracy and user friendliness of insulin pens in patients suffering from diabetes and related comorbidities. These parameters, along with patient introduction from prescribing physicians and level of training provided, drives patient pen selection and injection-regimen compliance to control their blood sugar., (© 2011 Diabetes Technology Society.)

Microelectrode array (MEA) technology holds tremendous potential in the fields of biodetection, lab-on-a-chip applications, and tissue engineering by facilitating noninvasive electrical interaction with cells in vitro. To date, significant efforts at integrating the cellular component with this detection technology have worked exclusively with neurons or cardiac myocytes. We investigate the feasibility of using MEAs to record from skeletal myotubes derived from primary myoblasts as a way of introducing a third electrogenic cell type and expanding the potential end applications for MEA-based biosensors. We find that the extracellular action potentials (EAPs) produced by spontaneously contractile myotubes have similar amplitudes to neuronal EAPs. It is possible to classify myotube EAPs by biological signal source using a shape-based spike sorting process similar to that used to analyze neural spike trains. Successful spike-sorting is indicated by a low within-unit variability of myotube EAPs. Additionally, myotube activity can cause simultaneous activation of multiple electrodes, in a similar fashion to the activation of electrodes by networks of neurons. The existence of multiple electrode activation patterns indicates the presence of several large, independent myotubes. The ability to identify these patterns suggests that MEAs may provide an electrophysiological basis for examining the process by which myotube independence is maintained despite rapid myoblast fusion during differentiation. Finally, it is possible to use the underlying electrodes to selectively stimulate individual myotubes without stimulating others nearby. Potential uses of skeletal myotubes grown on MEA substrates include lab-on-a-chip applications, tissue engineering, co-cultures with motor neurons, and neural interfaces., (Copyright © 2011 American Institute of Chemical Engineers (AIChE).)

The goal of this study is to assess whether the use of different aged blood, used during the simulated PennState Hershey Pediatric cardiopulmonary bypass (CPB)model affects immune activation. In order to study and compare the cytokine release involved in the humoral immune response during simulated CPB, both freshly drawn whole blood used less than 1 h after donation (n = 2)and reconstituted whole blood (1 week old) (n = 3) were circulated in a simulated CPB circuit under identical perfusion conditions. Discrete samples were collected in both experiments and analyzed for the proinflammatory cytokines concentrations of tumor necrosis factor-alpha, interleukin(IL)-6, and IL-8 using immunofluorocytometry as an indicator of immune activation. The results indicated that the cytokine concentrations of freshly drawn blood increased significantly compared with the reconstituted blood over the CPB circulation time. The fresh blood activation was two to three orders of magnitude larger than the week-old blood for all cytokines analyzed. These results suggest that the use of freshly drawn blood is required to evaluate immune responses to the extracorporeal circulation.

This article presents a microfluidic device which integrates autonomous serial immunofluorocytometry binding reactions of cytometric beads with fluorescence detection and quantification in a continuous flow environment. The microdevice assay is intended to alleviate the extensive benchwork and large sample volumes used when conducting traditional immunoassays, without requiring complex external controls. The technology is based on the miniaturization and automation of the serial processing steps of an antigen sandwich immunoassay, with integrated fluorescence detection using paramagnetic microbeads. The continuous flow design may enable temporal tracking of time-varying protein concentrations in a continuously infused sample for clinical applications, specifically for monitoring inflammation marker proteins in blood produced during cardiac surgeries involving cardiopulmonary bypass (CPB) procedures. The device operation was first validated via a single incubation device which measured the concentration of a fluorescently labeled biotin molecule using streptavidin-coated paramagnetic cytometric beads. Subsequently, a dual incubation device was tested with samples of the anaphylatoxin complement protein C3a, and was shown to be capable of differentiating between samples at typical systemic concentrations of the protein (1-5 mug/ml), with very low sample usage (<6 mul/h). It is believed that this continuous flow, automated microimmunosensor technology will be a platform for high sample rate immunoassays capable of tracking and more thoroughly characterizing the systemic inflammation process, and may aid in the development of better treatment options for systemic inflammation during and after CPB.

Skeletal muscle is the largest tissue in the body by weight and plays many roles in maintaining homeostasis and health. Ex vivo cell-based experimental systems used to study muscle cell contraction, and others based on incorporation of cells into sensitive force transducers or electrophysiology equipment, are time-consuming, invasive, and not universally available, slowing the pace of research. Video microscopy provides a noninvasive way to record the contractile behavior of skeletal muscle cells in vitro. We have developed a numerical procedure using image processing and pattern recognition algorithms, that makes it possible to quantify contractile behavior of multiple myotubes simultaneously, based on video data. We examined the ability of the program to identify movement using a simplified graphical model of myotube contraction and found that the program's success is dependent on the morphology and movement characteristics of the objects. However, the program performs optimally over the types of motions approximating those observed in culture and identifies contracting myotubes in sample videomicrographs of muscle cells in vitro. This program quantifies contractility on a population level, can be adapted for use in laboratories capable of digital video capture from a microscope, and may be coupled with other experimental techniques to supplement existing research tools., (2010 Wiley-Liss, Inc.)

A method for integrating porous polymer membranes such as polycarbonate, polyethersulfone and polyethylene terephthalate to microfluidic devices is described. The use of 3-aminopropyltriethoxysilane as a chemical crosslinking agent was extended to integrate membranes with PDMS and glass microfluidic channels. A strong, irreversible bond between the membranes and microfluidic structure was achieved. The bonding strength in the APTES treated devices was significantly greater than in devices fabricated using either a PDMS "glue" or two-part epoxy bonding method. Evaluation of a filtering microdevice and the pore structure via SEM indicates the APTES conjugation does not significantly alter the membrane transport function and pore morphology.

Background: The main concern in noninvasive (NI) glucose measurement is achieving high accuracy readings, although no blood (or other fluid) is involved in the process. Using methods based on different physical properties of a measured object can ensure the independence of each of the readings and therefore improve the validity of the end result. By using a combination of (three) independent technologies-ultrasonic, electromagnetic, and thermal-GlucoTrack presents a unique approach for a real-time, truly NI blood glucose spot measurement., Methods: Clinical trials were performed in two stages. Stage 1 was an initial method validation and performance verification of the device. In this stage, 50 type 1 and 2 diabetic patients, as well as healthy subjects, were evaluated with GlucoTrack against Ascensia Elite (Bayer). In the second stage, 85 additional diabetic subjects were evaluated in half and full daytime sessions using a GlucoTrack comparison with HemoCue (Glucose 201+)., Results: A total of 135 subjects were tested during the trial period, producing 793 data pairs. Using Clarke error grid analysis, 92% of the readings fell in the clinically acceptable zones A and B, with 50% in the A zone. Mean and median relative absolute differences were 29.9 and 19.9%, respectively., Conclusions: Integrating several modalities for NI assessment of glucose level enables more accurate readings, while a possible aberration in one modality is bypassed by the others. The present generation of GlucoTrack gives promising results; however, further improvement of the accuracy of the device is needed., ((c) 2009 Diabetes Technology Society.)

Cardiopulmonary bypass (CPB) circuits are frequently necessary in the repair of congenital heart defects in infants and children. Although advances in technology and operative technique have decreased the mortality associated with cardiac procedures requiring CPB, post-operative neuro-cognitive outcome and the role of the CPB circuit in post-operative morbidity remains a significant concern. There are several factors that have been suggested to play a significant role in general post-operative outcome, including intraoperative inflammatory responses caused by the interaction of blood with circuit component surfaces, selection of appropriate perfusion mode to optimize organ function during CPB, and the introduction of gaseous microemboli into the patient's systemic circulation through circuit manipulations and modifications. These factors are the subject of continuing research at the Penn State Hershey Children's Hospital Pediatric Cardiac Research Laboratories, and this review will focus on the results of studies aimed at identifying circuit elements that affect the delivery of gaseous microemboli to the patient during CPB procedures, the role of anti-factor D monoclonal antibody in reducing systemic inflammation during CPB, and the results of preliminary plasma proteomics studies conducted on infants undergoing CPB.

Microdialysis probes have been used for diabetes treatment as continuous monitoring system coupled to a glucose sensor. An on-chip microdialysis system with in-line sensing electrodes is demonstrated. As a first step towards greater biosensor integration with this miniaturized microdialysis system, a stacked system with in-line sensing electrodes was developed. Impedance electrodes sputtered within the microchannels were used to determine fluid electrical resistance from a dialyzed phosphate buffered saline (PBS) solution, which characterizes solution conductivity as a function of PBS concentration. The permeability of the membrane to the salt ions was obtained as 0.246+/-0.028 microm/s (15 nm pores). Subsequently, experiments measuring PBS dialysis in the time-domain at 64.4% recovery were conducted. The PBS concentration of the reservoir was changed in both a step response and sinusoidally with an 800 s period. The subsequently measured impedance indicates that the system is able to continuously track concentration changes in the reservoir with a 210 s system response delay. Most of this delay is due to the dead volume within the tubing between the syringe pumps and the microsystem. In addition, the predicted response was modeled using linear systems theory and matches the experimental measurements (r=0.98). This system is expected to have the proper sensitivity to track physiologically relevant concentration changes of biomolecules such as glucose (which has a physiological maximum change rate of approximately 4 mg/dl min with a periodicity of 1h or greater) with minimal lag time and amplitude reduction.

Background: Microdialysis is a sampling technique based on controlling the mass transfer rate of different-sized molecules across a semipermeable membrane. Because the dialysis process has minimal effects on the surrounding fluid, it is viewed as a tool for continuous monitoring of human metabolites. In diabetes treatment, microdialysis probes have been used as sampling systems coupled to a glucose biosensor but may struggle to obtain high recoveries of analytes, as the sampling housing, probes, and glucose sensors are fabricated as separate pieces and then assembled, resulting in a large dead volume, which limits sensing frequency. An in situ combination of a miniaturized microdialysis probe with an integrated glucose sensor could help solve some of these problems., Method: The system was fabricated by bonding a 6-mum-thick polycarbonate track-etch membrane with 100-nm-diameter pores onto microfluidic channels with the electrochemical glucose sensing electrodes patterned within the microchannels., Results: In vitro experiments demonstrating glucose microdialysis with continuous sensing were conducted. The permeability of glucose to the polycarbonate membrane with a 100-nm-diameter pore size was obtained to be 5.44 mum/s. Glucose recovery of 99% was observed using this microdialysis system at a perfusion flow rate of 0.5 microl/min. Experiments monitoring fluctuating glucose concentrations in the time domain at 99% recovery were also performed. The lag time was measured to be 210 seconds with 45 seconds contributed by mass transfer limitations and the rest from dead volume within the experimental setup., Conclusion: The electrochemical sensing component was able to continuously track concentration changes in the reservoir. This system is expected to have the proper sensitivity to track physiologically relevant concentration changes of glucose with a lag time of less than 1 minute and minimal amplitude reduction for continuous glucose monitoring for diabetes treatment.

A microfluidic device for continuous biosensing based on analyte binding with cytometric beads is introduced. The operating principle of the continuous biosensing is based on a novel concept named the "particle cross over" mechanism in microfluidic channels. By carefully designing the microfluidic network the beads are able to "cross-over" from a carrier fluid stream into a recipient fluid stream without mixing of the two streams and analyte dilution. After crossing over into the recipient stream, bead processing such as analyte-bead binding may occur. The microfluidic device is composed of a bead solution inlet, an analyte solution inlet, two washing solution inlets, and a fluorescence detection window. To achieve continuous particle cross over in microfluidic channels, each microfluidic channel is precisely designed to allow the particle cross over to occur by conducting a series of studies including an analogous electrical circuit study to find optimal fluidic resistances, an analytical determination of device dimensions, and a numerical simulation to verify microflow structures within the microfluidic channels. The functionality of the device was experimentally demonstrated using a commercially available fluorescent biotinylated fluorescein isothiocyanate (FITC) dye and streptavidin coated 8 microm-diameter beads. After, demonstrating particle cross over and biotin-streptavidin binding, the fluorescence intensity of the 8 microm-diameter beads was measured at the detection window and linearly depends on the concentration of the analyte (biotinylated FITC) at the inlet. The detection limit of the device was a concentration of 50 ng ml(-1) of biotinylated FITC.

As an extension of previous work, a microfluidic device, which can separate blood plasma in a continuous, real-time fashion from a whole blood, is successfully integrated with a mock cardiopulmonary bypass circuit. The functionality of the device is demonstrated with the use of freshly harvested bovine blood. The plasma selectivities were 100% and 99.4% and the plasma separation volume percents were 18.7% and 24.5% for 26% and 37% inlet hematocrit levels, respectively. As an advanced stage of this research, a microfluidic device, which can measure the concentration of clinically relevant blood plasma protein in a continuous fashion, is being developed on the basis of fluid handling circuits coupled to fluorescent cytometric bead assays. The functionality of the device is demonstrated with the use of a biotinylated FITC solution and a streptavidin-coated, 8-mum-diameter bead. The binding event between biotinylated FITC and the streptavidin bead is continuously detected within a detection window at the outlet of the device. For a known concentration (1 microg/ml) of biotinylated FITC solution, the measured fluorescent intensity is fairly constant and shows a stable gaussian distribution of the bead fluorescence intensity. It is expected that the proposed device can be used for continuous measurement of clinically relevant proteins during cardiac surgery with the cardiopulmonary bypass procedure.