A Regenerable Biosensing Platform for Bacterial Toxins

Waterborne diarrheal diseases such as travelers’ diarrhea and cholera remain a threat to public health in many countries. Rapid diagnosis of an infectious disease is critical in preventing the escalation of a disease outbreak into an epidemic. Many of the diagnostic tools for infectious diseases employed today are time-consuming and require specialized laboratory settings and trained personnel. There is hence a pressing need for fit-for-purpose point-of-care diagnostic tools with emphasis in sensitivity, specificity, portability, and low cost. We report work toward thermally reversible biosensors for detection of the carbohydrate-binding domain of the Escherichia coli heat-labile enterotoxin (LTB), a toxin produced by enterotoxigenic E. coli strains, which causes travelers’ diarrhea. The biosensing platform is a hybrid of two materials, combining the optical properties of porous silicon (pSi) interferometric transducers and a thermoresponsive multivalent glycopolymer, to enable recognition of LTB. Analytical performance of our biosensors allows us to detect, using a label-free format, sub-micromolar concentrations of LTB in solution as low as 0.135 μM. Furthermore, our platform shows a temperature-mediated “catch-and-release” behavior, an exciting feature with potential for selective protein capture, multiple readouts, and regeneration of the sensor over consecutive cycles of use

Chemotaxis of Cell Populations through Confined Spaces at Single-Cell Resolution

Cell migration is crucial for both physiological and pathological processes. Current in vitro cell motility assays suffer from various drawbacks, including insufficient temporal and/or optical resolution, or the failure to include a controlled chemotactic stimulus. Here, we address these limitations with a migration chamber that utilizes a self-sustaining chemotactic gradient to induce locomotion through confined environments that emulate physiological settings. Dynamic real-time analysis of both population-scale and single-cell movement are achieved at high resolution. Interior surfaces can be functionalized through adsorption of extracellular matrix components, and pharmacological agents can be administered to cells directly, or indirectly through the chemotactic reservoir. Direct comparison of multiple cell types can be achieved in a single enclosed system to compare inherent migratory potentials. Our novel microfluidic design is therefore a powerful tool for the study of cellular chemotaxis, and is suitable for a wide range of biological and biomedical applications

Influence of Surface Ligand Density and Particle Size on the Penetration of the Blood–Brain Barrier by Porous Silicon Nanoparticles

Overcoming the blood–brain barrier (BBB) remains a significant challenge with regard to drug delivery to the brain. By incorporating targeting ligands, and by carefully adjusting particle sizes, nanocarriers can be customized to improve drug delivery. Among these targeting ligands, transferrin stands out due to the high expression level of its receptor (i.e., transferrin receptor) on the BBB. Porous silicon nanoparticles (pSiNPs) are a promising drug nanocarrier to the brain due to their biodegradability, biocompatibility, and exceptional drug-loading capacity. However, an in-depth understanding of the optimal nanoparticle size and transferrin surface density, in order to maximize BBB penetration, is still lacking. To address this gap, a diverse library of pSiNPs was synthesized using bifunctional poly(ethylene glycol) linkers with methoxy or/and carboxyl terminal groups. These variations allowed us to explore different transferrin surface densities in addition to particle sizes. The effects of these parameters on the cellular association, uptake, and transcytosis in immortalized human brain microvascular endothelial cells (hCMEC/D3) were investigated using multiple in vitro systems of increasing degrees of complexity. These systems included the following: a 2D cell culture, a static Transwell model, and a dynamic BBB-on-a-chip model. Our results revealed the significant impact of both the ligand surface density and size of pSiNPs on their ability to penetrate the BBB, wherein intermediate-level transferrin densities and smaller pSiNPs exhibited the highest BBB transportation efficiency in vitro. Moreover, notable discrepancies emerged between the tested in vitro assays, further emphasizing the necessity of using more physiologically relevant assays, such as a microfluidic BBB-on-a-chip model, for nanocarrier testing and evaluation

Crossed flow microfluidics for high throughput screening of bioactive chemical–cell interactions

This paper describes the use of crossed laminar flow microfluidics for the selective capture of multiple cell types on-chip aiming for high throughput screening of various cell treatment compounds. Parallel laminar streams containing different cell types were perfused and captured on a cell adhesion protein-functionalized reaction area. Thereafter, parallel streams containing cell treatment solutions were delivered orthogonally over the captured cells. Multiple cell types and a range of cell treatment conditions could therefore be assessed in a single experiment. We were also able to sort mixed cell populations via antibody array clusters, and to further deliver treatments to subpopulations of cells. Moreover, using solutions with different tonicities, we successfully demonstrated the incorporation of a live/dead cell viability assessment on-chip for a direct read out assay following the treatments. This crossed laminar flow microfluidics for generation of a cell-based assay could therefore offer an interesting platform for high throughput screening of potential drug candidates, nanoparticle toxicity testing, or other cellular and molecular interventions

The Use of Microfluidics in Cytotoxicity and Nanotoxicity Experiments

Many unique chemical compounds and nanomaterials are being developed, and each one requires a considerable range of in vitro and/or in vivo toxicity screening in order to evaluate their safety. The current methodology of in vitro toxicological screening on cells is based on well-plate assays that require time-consuming manual handling or expensive automation to gather enough meaningful toxicology data. Cost reduction; access to faster, more comprehensive toxicity data; and a robust platform capable of quantitative testing, will be essential in evaluating the safety of new chemicals and nanomaterials, and, at the same time, in securing the confidence of regulators and end-users. Microfluidic chips offer an alternative platform for toxicity screening that has the potential to transform both the rates and efficiency of nanomaterial testing, as reviewed here. The inherent advantages of microfluidic technologies offer high-throughput screening with small volumes of analytes, parallel analyses, and low-cost fabrication

Direct comparison of multiple cell types in an enclosed migration system.

<p>(a) A representative image of mixture of MCF-7 and MDA-MB-2321 cells migrating through 20 µm and 10 µm wide microchannels. A surface map of fluorescence intensity (b) and horizontal intensity profile (c) are shown as adiditonal methods for quantifying the extent of migration for each cell type (horizontal axis of panel (c) drawn to scale to correspond with cell position in panel (a)).</p

The influence of channel width on migratory cell morphology and migration speed.

<p>HOS cells are pictured as they migrate ‘upward’ toward a chemokine source through microchannels of various widths. Cells migrating in 50 µm (a) and 20 µm (b) -wide channels exhibit morphologies similar to cells on a planar surface (f). Cells migrating through 10 µm (c) and 6 µm (d) channels are laterally restricted by the channel walls. Cells migrating through 3 µm-wide channels (e) undergo significant deformation, becoming uniform and morphologically de-polarized. Scale bar for (a–f) is indicated in panel (f) as 50 µm. g) Cell migration speed inside different channels of each dimension was calculated, and shown to be influenced by microchannel width (error bars depict standard error of the mean).</p