a b s t r a c tA layer-by-layer (LbL) strategy of modifying an electrode with a specific combination of polymeric and xerogel materials is used to create an effective first generation biosensor for uric acid (UA) -a clinically relevant molecule implemented in pregnancy induced hypertension (PIH) diagnosis, a condition that can lead to a serious disorder called preeclampsia. In addition to offering a new, promising sensor for UA, this study represents significant progress for amperometric biosensor development since the strategy and materials employed were successfully and readily adapted from a glucose biosensor model scheme and used for the effective detection of UA, a fundamentally different molecule compared to glucose. Specifically, each of four, functional modifying layers (outer polyurethane (PU) selective membrane, the inner selective electropolymer, and the xerogel bi-layer) are systematically investigated and tailored for UA permeability and interferent discrimination. The role of PU hydrophobicity and its UA permeability are established while the enzyme-doped and outer diffusional xerogel layers are evaluated for uricase (UOx) species/loading and silane precursor dependence, respectively. LbL systematic evaluation reveals the specific combination of hydroxyl-methyl triethoxy silane (HMTES) xerogels, a polyluminol-aniline electropolymer, and 100% hydrophilic polyurethane yields impressive uric acid sensing performance: effective sensitivity (0.8 nA/μM), linear response across physiologically relevant UA concentrations (100-700 μM), fast response times (~10 s), low limits of detection (b 10 μM), and selectivity against most common interferents. Toward the specific application of PIH risk assessment, the optimized sensor exhibited 10 day stability as well as effective shelf-life exceeding 35 days. The presented system rivals or exceeds UA biosensor performance found in the literature and offers the possibility of miniaturization for in situ or in vivo remote diagnostic sensing. The successful adaptation suggests that the strategy and materials may be applicable to detecting/monitoring other medically significant molecules via sensor development.

Biosensing strategies that employ readily adaptable materials for different analytes, can be miniaturized into needle electrode form, and function in bodily fluids represent a significant step toward the development of clinically relevant in vitro and in vivo sensors. In this work, a general scheme for 1st generation amperometric biosensors involving layer-by-layer electrode modification with enzyme-doped xerogels, electrochemically-deposited polymer, and polyurethane semi-permeable membranes is shown to achieve these goals. With minor modifications to these materials, sensors representing potential point-of-care medical tools are demonstrated to be sensitive and selective for a number of conditions. The potential for bedside measurements or continuous monitoring of analytes may offer faster and more accurate clinical diagnoses for diseases such as diabetes (glucose), preeclampsia (uric acid), galactosemia (galactose), xanthinuria (xanthine), and sepsis (lactate). For the specific diagnostic application, the sensing schemes have been miniaturized to wire electrodes and/or demonstrated as functional in synthetic urine or blood serum. Signal enhancement through the incorporation of platinum nanoparticle film in the scheme offers additional design control within the sensing scheme. The presented sensing strategy has the potential to be applied to any disease that has a related biomolecule and corresponding oxidase enzyme and represents rare, adaptable, sensing capabilities.

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Ultrasound-targeted microbubble cavitation (UTMC) transiently opens the blood brain barrier (BBB). We previously determined that UTMC induces BBB hyperpermeability through an influx of calcium. As activation of RhoA is a calcium-dependent pathway that causes cytoskeletal reorganization, leading to the breakdown of tight junctions, we tested the hypothesis that UTMC-induced activation of RhoA leads to BBB hyperpermeability. We utilized a transwell model with brain endothelial cells and astrocytes on opposite sides of a support membrane. Ultrasound (1 MHz, 250 kPa, 10 μs pulse duration, 10 ms pulse interval) was applied in the presence of lipid microbubbles for 20 s. BBB permeability was assessed using dextran flux and transendothelial electrical resistance (TEER) measured across the membrane. Integrity of tight junctions was evaluated by staining for ZO-1. UTMC reduced TEER (p < 0.05), confirming reduced barrier integrity. One hour after UTMC, there was a significant decrease in ZO-1 mean pixel intensity (p < 0.05). Treatment of cells with Rho inhibitor II (Y16) significantly reduced UTMC-induced dextran flux across the BBB (p < 0.05) and UTMC-induced decrease in TEER. In our co-culture model of the BBB, UTMC induces hyperpermeability through modulation of tight junctions, at least in part through a RhoA-dependent mechanism.

Introduction: Stem cell and AAV-gene therapies are exciting new treatments for neuronal regeneration after ischemic stroke, but they have yet to become the standard of care. One challenge for these therapies is penetrating the blood brain barrier ( BBB ). Ultrasound-targeted microbubble cavitation ( UTMC ) using contrast agents (microbubbles, MB ) and ultrasound ( US ) applied to the brain is being explored to transiently open the BBB. While UTMC-mediated BBB opening is a promising delivery strategy, its underlying mechanisms are poorly understood. We hypothesize that tight junctions ( TJ ) play a role in mediating BBB permeability after UTMC. Methods: We utilized transwells with murine brain endothelial cells ( EC ) and astrocytes on opposite sides of a support membrane. US (1 MHz, 10 μs duration, 10 ms pulse interval) at 250 kPa was applied for 20 sec to MB in contact with ECs. Permeability was assessed using dextran. Immunofluorescence with staining for CD-31 (EC) and ZO-1 (TJ) was performed on transwells fixed 15 and 60 min after UTMC. Z-stacks of the transwells were acquired via confocal microscopy to measure paracellular gap area and mean voxel intensity of ZO-1. Data was normalized per EC. Unpaired 2-tailed t -test with correction for multiple t-tests were performed. Results: UTMC increases dextran flux across the BBB (p<0.05). As shown in Figure 1, compared to no UTMC, there is an increase in paracellular gap area per cell (p<0.05). From 15 to 60 min after UTMC, there is a decrease in gap area (p=0.056) and an increase in the mean voxel intensity of ZO-1 per EC (p<0.05). Conclusions: UTMC increase BBB permeability through paracellular gaps. Between 15 and 60 min after UTMC, there is closure of some paracellular gaps and a re-establishment of endothelial TJ in this model of the BBB. Understanding the mechanisms mediating the changes in the BBB after UTMC should facilitate the development of UTMC as a delivery strategy for cell or gene-based therapeutics to enhance recovery after stroke.