Nanoporous Hydrogels for the Observation of Anthrax Exotoxin Translocation Dynamics.

The ability to observe lethal anthrax exotoxins translocating through size-constricting nanopores in vitro, combined with detailed sequence and structural data, has aided in elucidated mechanisms of exotoxin cell entry and toxicity. However, due to limited observations of anthrax exotoxins translocating through protective antigen nanopores in vitro and the instability of protective antigen-functionalized suspended lipid bilayers, questions remain regarding the native mechanisms of cell entry. Nanoporous hydrogel membranes offer a robust tool for studying protein translocation with ensemble measurements that complement conventional single-molecule translocation measurements.

A Reversibly Sealed, Easy Access, Modular (SEAM) Microfluidic Architecture to Establish In Vitro Tissue Interfaces.

Microfluidic barrier tissue models have emerged as advanced in vitro tools to explore interactions with external stimuli such as drug candidates, pathogens, or toxins. However, the procedures required to establish and maintain these systems can be challenging to implement for end users, particularly those without significant in-house engineering expertise. Here we present a module-based approach that provides an easy-to-use workflow to establish, maintain, and analyze microscale tissue constructs.

A Genome-Wide RNA Interference Screen Identifies a Role for Wnt/β-Catenin Signaling during Rift Valley Fever Virus Infection.

Unlabelled: Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered.

An embedded microretroreflector-based microfluidic immunoassay platform.

We present a microfluidic immunoassay platform based on the use of linear microretroreflectors embedded in a transparent polymer layer as an optical sensing surface, and micron-sized magnetic particles as light-blocking labels. Retroreflectors return light directly to its source and are highly detectable using inexpensive optics. The analyte is immuno-magnetically pre-concentrated from a sample and then captured on an antibody-modified microfluidic substrate comprised of embedded microretroreflectors, thereby blocking reflected light.

Spatiotemporal pH dynamics in concentration polarization near ion-selective membranes.

We present a detailed analysis of the transient pH dynamics for a weak, buffered electrolyte subject to voltage-driven transport through an ion-selective membrane. We show that pH fronts emanate from the concentration polarization zone next to the membrane and that these propagating fronts change the pH in the system several units from its equilibrium value. The analysis is based on a 1D model using the unsteady Poisson-Nernst-Planck equations with nonequilibrium chemistry and without assumptions of electroneutrality or asymptotically thin electric double layers.

Rapid detection of trace bacteria in biofluids using porous monoliths in microchannels.

We present advancements in microfluidic technology for rapid detection of as few as 10 rickettsial organisms in complex biological samples. An immuno-reactive filter, macroporous polyacrylamide monolith (PAM), fabricated within a microfluidic channel enhances solid-phase immuno-capture, staining and detection of targeted bacteria. Bacterial cells in samples flowing through the channel are forced to interact with the PAM filter surface due to size exclusion, overcoming common transport and kinetic limitations for rapid (min), high-efficiency (~100%) capture.

Thirty-minute total synthesis of microfluidic systems and functionalized porous elements via "living" radical photo-polymerization.

A "living" radical photo-polymerization (LRPP) technique is used to rapidly fabricate microfluidic channels and micro-patterned porous polymer monoliths. Surface-initiated LRPP is then used to functionalize porous elements in a robust one-step surface modification process. Assay-ready platforms can be fully realized in less than 30 minutes.

Spatiotemporal mapping of concentration polarization induced pH changes at nanoconstrictions.

Under an applied electric field, concentration polarization (CP) arises from ion permselectivity of most nanoporous materials and biological ion channels. We present novel methods to quantitatively assess CP-induced spatiotemporal changes of pH that may significantly impact transport dynamics, device functionality, and physicochemical properties of molecular analytes in devices with nanofluidic constrictions. We measured pH fluctuations of >1.

Microfluidic digital isoelectric fractionation for rapid multidimensional glycoprotein analysis.

Here we present an integrated microfluidic device for rapid and automated isolation and quantification of glycoprotein biomarkers directly from biological samples on a multidimensional analysis platform. In the first dimension, digital isoelectric fractionation (dIEF) uses discrete pH-specific membranes to separate proteins and their isoforms into precise bins in a highly flexible spatial arrangement on-chip. dIEF provides high sample preconcentration factors followed by immediate high-fidelity transfer of fractions for downstream analysis.

Microscale isoelectric fractionation using photopolymerized membranes.

In this work, we introduce microscale isoelectric fractionation (μIF) for isolation and enrichment of molecular species at any desired location in a microfluidic chip. Narrow pH-specific polyacrylamide membranes are photopatterned in situ for customizable device fabrication; multiple membranes of precise pH are easily incorporated throughout existing channel layouts. Samples are electrophoretically driven across the membranes such that charged species, for example, proteins and peptides, are rapidly discretized into fractions based on their isoelectric points (pI) without the use of carrier ampholytes.

Aptamers as affinity reagents in an integrated electrophoretic lab-on-a-chip platform.

Nucleic acid based affinity reagents (e.g., aptamers) offer several possible advantages over antibodies as specific recognition elements in biochemical assays.

Enrichment and fractionation of proteins via microscale pore limit electrophoresis.

In this work we photopolymerized precise and well-controlled polyacrylamide porosity gradients in microchannels for microscale pore limit electrophoresis (microPLE) of proteins. Porosity was controlled via distributions of acrylamide monomer and bisacrylamide crosslinker. MicroPLE provides high-resolution fractionation of complex samples based on the spatial dependence of each species' electrophoretic pore limit--the porosity at which a protein's electrophoretic mobility is negligible due to its molecular size.

IEF in microfluidic devices.

IEF is one of the most powerful and prevalent techniques used in separation sciences. The power of IEF comes from the fact that it not only separates analytes based on their pI but also focuses them into highly resolved bands. In line with the miniaturization trend spurring the analytical community, the past decade has yielded a wealth of research focused on implementing IEF in microfluidic chip-based formats (microIEF).

An integrated microfluidic platform for sensitive and rapid detection of biological toxins.

Towards designing a portable diagnostic device for detecting biological toxins in bodily fluids, we have developed microfluidic chip-based immunoassays that are rapid (< 20 minutes), require minimal sample volume (<10 microL) and have appreciable sensitivity and dynamic range (microM-pM). The microfluidic chip is being integrated with miniaturized electronics, optical elements, fluid-handling components, and data acquisition software to develop a portable, self-contained device. The device is intended for rapid, point-of-care (and, in future, point-of-incident) testing in case of an accidental or intentional exposure/intoxication to biotoxins.

On-chip isoelectric focusing using photopolymerized immobilized pH gradients.

We present the first successful adaptation of immobilized pH gradients (IPGs) to the microscale (muIPGs) using a new method for generating precisely defined polymer gradients on-chip. Gradients of monomer were established via diffusion along 6 mm flow-restricted channel segments. Precise control over boundary conditions and the resulting gradient is achieved by continuous flow of stock solutions through side channels flanking the gradient segment.

Integrated microfluidic platform for oral diagnostics.

While many point-of-care (POC) diagnostic methods have been developed for blood-borne analytes, development of saliva-based POC diagnostics is in its infancy. We have developed a portable microfluidic device for detection of potential biomarkers of periodontal disease in saliva. The device performs rapid microfluidic chip-based immunoassays (<3-10 min) with low sample volume requirements (10 microL) and appreciable sensitivity (nM-pM).

Microfluidic immunoassays as rapid saliva-based clinical diagnostics.

At present, point-of-care (POC) diagnostics typically provide a binary indication of health status (e.g., home pregnancy test strip).

Integrated preconcentration SDS-PAGE of proteins in microchips using photopatterned cross-linked polyacrylamide gels.

The potential of integration of functions in microfluidic chips is demonstrated by implementing on-chip preconcentration of proteins prior to on-chip protein sizing by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two polymeric elements-a thin (approximately 50 microm) size exclusion membrane for preconcentration and a longer (approximately cm) porous monolith for protein sizing-were fabricated in situ using photopolymerization. Contiguous placement of the two polymeric elements in the channels of a microchip enabled simple and zero dead volume integration of the preconcentration with SDS-PAGE.

Controlled microfluidic reconstitution of functional protein from an anhydrous storage depot.

A novel method has been developed for preserving molecules in microfluidic devices that also enables the control of the spatial and temporal concentrations of the reconstituted molecules within the devices. In this method, a storage cavity, embedded in a microchannel, is filled with a carbohydrate matrix containing, for example, a reagent. When the matrix is exposed to flowing liquid, it dissolves, resulting in the controlled reconstitution and release of the reagent from the cavity.