Hansang Cho

Label-free and highly sensitive biomolecular detection using SERS and electrokinetic preconcentration

In this paper, we present a method combining surface-enhanced Raman scattering (SERS) spectroscopy to detect biomolecules in a label-free way with an electrokinetic preconcentration technique (electrophoresis) to amplify biomolecular signals at low concentrations. A constant electric field is applied to charged biomolecules in solution, attracting them to an oppositely charged electrode, which is also used as a SERS substrate. Within 5 min, we observed that the SERS signal of 10 fM adenine was amplified to the level of the signal of non-preconcentrated 1 microM adenine (sensitivity improvement by 8 orders of magnitude) and the method was effective over a wide range of concentrations (10 fM to 1 microM). The signals were further amplified under stronger electric field and longer application: The increase of the signal intensity was observed to be 51 times at -0.6 V cm(-1) after 25 min. The effectiveness of this method allows the creation of label-free, target-specific, and highly sensitive monitoring applications.

Neuronal uptake and propagation of a rare phosphorylated high-molecular-weight tau derived from Alzheimer’s disease brain

Tau pathology is known to spread in a hierarchical pattern in Alzheimers disease (AD) brain during disease progression, likely by trans-synaptic tau transfer between neurons. However, the tau species involved in inter-neuron propagation remains unclear. To identify tau species responsible for propagation, we examined uptake and propagation properties of different tau species derived from postmortem cortical extracts and brain interstitial fluid of tau-transgenic mice, as well as human AD cortices. Here we show that PBS-soluble phosphorylated high-molecular-weight (HMW) tau, though very low in abundance, is taken up, axonally transported, and passed on to synaptically connected neurons. Our findings suggest that a rare species of soluble phosphorylated HMW tau is the endogenous form of tau involved in propagation and could be a target for therapeutic intervention and biomarker development.

Three-Dimensional Blood-Brain Barrier Model for in vitro Studies of Neurovascular Pathology

Blood–brain barrier (BBB) pathology leads to neurovascular disorders and is an important target for therapies. However, the study of BBB pathology is difficult in the absence of models that are simple and relevant. In vivo animal models are highly relevant, however they are hampered by complex, multi-cellular interactions that are difficult to decouple. In vitro models of BBB are simpler, however they have limited functionality and relevance to disease processes. To address these limitations, we developed a 3-dimensional (3D) model of BBB on a microfluidic platform. We verified the tightness of the BBB by showing its ability to reduce the leakage of dyes and to block the transmigration of immune cells towards chemoattractants. Moreover, we verified the localization at endothelial cell boundaries of ZO-1 and VE-Cadherin, two components of tight and adherens junctions. To validate the functionality of the BBB model, we probed its disruption by neuro-inflammation mediators and ischemic conditions and measured the protective function of antioxidant and ROCK-inhibitor treatments. Overall, our 3D BBB model provides a robust platform, adequate for detailed functional studies of BBB and for the screening of BBB-targeting drugs in neurological diseases.

Migration of neutrophils targeting amyloid plaques in Alzheimer's disease mouse model

Immune responses in the brain are thought to play a role in disorders of the central nervous system, but an understanding of the process underlying how immune cells get into the brain and their fate there remains unclear. In this study, we used a 2-photon microscopy to reveal that neutrophils infiltrate brain and migrate toward amyloid plaques in a mouse model of Alzheimers disease. These findings suggest a new molecular process underlying the pathophysiology of Alzheimers disease.

Microfluidic Chemotaxis Platform for Differentiating the Roles of Soluble and Bound Amyloid-β on Microglial Accumulation

Progressive microglial accumulation at amyloid-β (Aβ) plaques is a well-established signature of the pathology of Alzheimers disease, but how and why microglia accumulate in the vicinity of Aβ plaques is unknown. To understand the distinct roles of Aβ on microglial accumulation, we quantified microglial responses to week-long lasting gradients of soluble Aβ and patterns of surface-bound Aβ in microfluidic chemotaxis platforms. We found that human microglia chemotaxis in gradients of soluble Aβ42 was most effective at two distinct concentrations of 23 pg.mL−1 and 23 ng.mL−1 Aβ42 in monomers and oligomers. We uncovered that while the chemotaxis at higher Aβ concentrations was exclusively due to Aβ gradients, chemotaxis at lower concentrations was enhanced by Aβ-induced microglial production of MCP-1. Microglial migration was inhibited by surface-bound Aβ42 in oligomers and fibrils above 45 pg.mm−2. Better understanding of microglial migration can provide insights into the pathophysiology of senile plaques in AD.

A Food and Drug Administration-approved Asthma Therapeutic Agent Impacts Amyloid β in the Brain in a Transgenic Model of Alzheimer Disease

Background: Cromolyn sodium is an FDA-approved drug structurally similar to fisetin, an antiamyloidogenic molecule. Results: Cromolyn sodium interferes with amyloid β (Aβ) aggregation in vitro while rapidly decreasing the levels of soluble Aβ peptides in vivo after a week. Conclusion: Cromolyn sodium may have an impact on amyloid economy. Significance: Developing new disease-modifying therapeutics remains an urgent need in the treatment of Alzheimer disease. Interfering with the assembly of Amyloid β (Aβ) peptides from monomer to oligomeric species and fibrils or promoting their clearance from the brain are targets of anti-Aβ-directed therapies in Alzheimer disease. Here we demonstrate that cromolyn sodium (disodium cromoglycate), a Food and Drug Administration-approved drug already in use for the treatment of asthma, efficiently inhibits the aggregation of Aβ monomers into higher-order oligomers and fibrils in vitro without affecting Aβ production. In vivo, the levels of soluble Aβ are decreased by over 50% after only 1 week of daily intraperitoneally administered cromolyn sodium. Additional in vivo microdialysis studies also show that this compound decreases the half-life of soluble Aβ in the brain. These data suggest a clear effect of a peripherally administered, Food and Drug Administration-approved medication on Aβ economy, supporting further investigation of the potential long-term efficacy of cromolyn sodium in Alzheimer disease.

Microscale arrays for the profiling of start and stop signals coordinating human-neutrophil swarming

Neutrophil swarms protect healthy tissues by sealing off sites of infection. In the absence of swarming, microbial invasion of surrounding tissues can result in severe infections. Recent observations in animal models have shown that swarming requires rapid neutrophil responses and well-choreographed neutrophil migration patterns. However, in animal models physical access to the molecular signals coordinating neutrophil activities during swarming is limited. Here, we report the development and validation of large microscale arrays of zymosan-particle clusters for the study of human neutrophils during swarming ex vivo. We characterized the synchronized swarming of human neutrophils under the guidance of neutrophil-released chemokines, and measured the mediators released at different phases of human-neutrophil swarming against targets simulating infections. We found that the network of mediators coordinating human-neutrophil swarming includes start and stop signals, proteolytic enzymes and enzyme inhibitors, as well as modulators of activation of other immune and non-immune cells. We also show that the swarming behavior of neutrophils from patients following major trauma is deficient and gives rise to smaller swarms than those of neutrophils from healthy individuals.

Biologically inspired porous cooling membrane using arrayed-droplets evaporation

Inspired by human skin that dissipates heat via sweat-droplet evaporation on the skin, we propose an evaporative cooling technique via arrayed-droplets on a porous membrane, construct an analytical model of the evaporative cooling including interactions among the large number of droplets, and extensively analyze the cooling performance of 1000-droplet array. Our model shows that heat dissipation is enhanced by the membrane with dense droplets (i.e., enhancement factor of 10 at 5000 pores/cm2 and pore radius of 35 μm with respect to human skin). Additionally, the model predicts that the membrane dissipates heat more efficiently by operating at a higher temperature (i.e., additional enhancement factor of 17 at droplet temperature of 100 °C).

Synthesis of Cell-Adhesive Anisotropic Multifunctional Particles by Stop Flow Lithography and Streptavidin–Biotin Interactions

Cell-adhesive particles are of significant interest in biotechnology, the bioengineering of complex tissues, and biomedical research. Their applications range from platforms to increase the efficiency of anchorage-dependent cell culture to building blocks to loading cells in heterogeneous structures to clonal-population growth monitoring to cell sorting. Although useful, currently available cell-adhesive particles can accommodate only homogeneous cell culture. Here, we report the design of anisotropic hydrogel microparticles with tunable cell-adhesive regions as first step toward micropatterned cell cultures on particles. We employed stop flow lithography (SFL), the coupling reaction between amine and N-hydroxysuccinimide (NHS) and streptavidin-biotin chemistry to adjust the localization of conjugated collagen and poly-L-lysine on the surface of microscale particles. Using the new particles, we demonstrate the attachment and formation of tight junctions between brain endothelial cells. We also demonstrate the geometric patterning of breast cancer cells on particles with heterogeneous collagen coatings. This new approach avoids the exposure of cells to potentially toxic photoinitiators and ultraviolet light and decouples in time the microparticle synthesis and the cell culture steps to take advantage of the most recent advances in cell patterning available for traditional culture substrates.

On-demand, competing gradient arrays for neutrophil chemotaxis

Neutrophils are the most abundant type of white blood cells in the circulation, protecting the body against pathogens and responding early to inflammation. Although we understand how neutrophils respond to individual stimuli, we know less about how they prioritize between competing signals or respond to combinational signals. This situation is due in part to the lack of adequate experimental systems to provide signals in controlled spatial and temporal fashion. To address these limitations, we designed a platform for generating on-demand, competing chemical gradients and for monitoring neutrophil migration. On this platform, we implemented forty-eight assays generating independent gradients and employed synchronized valves to control the timing of these gradients. We observed faster activation of neutrophils in response to fMLP than to LTB4 and unveiled for the first time a potentiating effect for fMLP during migration towards LTB4. Our observations, enabled by the new tools, challenge the current paradigm of inhibitory competition between distinct chemoattractant gradients and suggest that human neutrophils are capable of complex integration of chemical signals in their environment.