Centrifugal microfluidic system for colorimetric sample-to-answer detection of viral pathogens.

We describe a microfluidic system for conducting thermal lysis, polymerase chain reaction (PCR) amplification, hybridization, and colorimetric detection of foodborne viral organisms in a sample-to-answer format. The on-chip protocol entails 24 steps which are conducted by a centrifugal platform that allows for actuating liquids pneumatically during rotation and so facilitates automation of the workflow. The microfluidic cartridge is fabricated from transparent thermoplastic polymers and accommodates assay components along with an embedded micropillar array for detection and read-out. A panel of oligonucleotide primers and probes has been developed to perform PCR and hybridization assays that allows for identification of five different viruses, including pathogens such as norovirus and hepatitis A virus (HAV) in a multiplexed format using digoxigenin-labelled amplicons and immunoenzymatic conversion of a chromogenic substrate. Using endpoint detection, we demonstrate that the system can accurately and repetitively (n = 3) discriminate positive and negative signals for HAV at 350 genome copies per µL. As part of the characterization and optimization process, we show that the implementation of multiple (e.g., seven) micropillar arrays in a narrow fluidic pathway can lead to variation (up to 50% or more) in the distribution of colorimetric signal deriving from the assay. Numerical modeling of flow behaviour was used to substantiate these findings. The technology-by virtue of automation-can provide a pathway toward rapid detection of viral pathogens, shortening response time in food safety surveillance, compliance, and enforcement as well as outbreak investigations.

Automated centrifugal microfluidic system for the preparation of adaptor-ligated sequencing libraries.

The intensive workload associated with the preparation of high-quality DNA libraries remains a key obstacle toward widespread deployment of sequencing technologies in remote and resource-limited areas. We describe the development of single-use microfluidic devices driven by an advanced pneumatic centrifugal microfluidic platform, the PowerBlade, to automate the preparation of Illumina-compatible libraries based on adaptor ligation methodology. The developed on-chip workflow includes enzymatic DNA fragmentation coupled to end-repair, adaptor ligation, first DNA cleanup, PCR amplification, and second DNA cleanup. This complex workflow was successfully integrated into simple thermoplastic microfluidic devices that are amenable to mass production with injection molding. The system was validated by preparing, on chip, libraries from a mixture of genomic DNA extracted from three common foodborne pathogens (Listeria monocytogenes, Escherichia coli and Salmonella enterica serovar Typhimurium) and comparing them with libraries made via a manual procedure. The two types of libraries were found to exhibit similar quality control metrics (including genome coverage, assembly, and relative abundances) and led to nearly uniform coverage independent of GC content. This microfluidic technology offers a time-saving and cost-effective alternative to manual procedures and robotic-based automation, making it suitable for deployment in remote environments where technical expertise and resources might be scarce. Specifically, it facilitates field practices that involve mid- to low-throughput sequencing, such as tasks related to foodborne pathogen detection, characterization, and microbial profiling.

Microstructured human fibroblast-derived extracellular matrix scaffold for vascular media fabrication.

In the clinical and pharmacological fields, there is a need for the production of tissue-engineered small-diameter blood vessels. We have demonstrated previously that the extracellular matrix (ECM) produced by fibroblasts can be used as a scaffold to support three-dimensional (3D) growth of another cell type. Thus, a resistant tissue-engineered vascular media can be produced when such scaffolds are used to culture smooth muscle cells (SMCs). The present study was designed to develop an anisotropic fibroblastic ECM sheet that could replicate the physiological architecture of blood vessels after being assembled into a small diameter vascular conduit. Anisotropic ECM scaffolds were produced using human dermal fibroblasts, grown on a microfabricated substrate with a specific topography, which led to cell alignment and unidirectional ECM assembly. Following their devitalization, the scaffolds were seeded with SMCs. These cells elongated and migrated in a single direction, following a specific angle relative to the direction of the aligned fibroblastic ECM. Their resultant ECM stained for collagen I and III and elastin, and the cells expressed SMC differentiation markers. Seven days after SMCs seeding, the sheets were rolled around a mandrel to form a tissue-engineered vascular media. The resulting anisotropic ECM and cell alignment induced an increase in the mechanical strength and vascular reactivity in the circumferential direction as compared to unaligned constructs. Copyright © 2016 John Wiley & Sons, Ltd.

3D thermoplastic elastomer microfluidic devices for biological probe immobilization.

Microfluidics has emerged as a valuable tool for the high-resolution patterning of biological probes on solid supports. Yet, its widespread adoption as a universal biological immobilization tool is still limited by several technical challenges, particularly for the patterning of isolated spots using three-dimensional (3D) channel networks. A key limitation arises from the difficulties to adapt the techniques and materials typically used in prototyping to low-cost mass-production. In this paper, we present the fabrication of thin thermoplastic elastomer membranes with microscopic through-holes using a hot-embossing process that is compatible with high-throughput manufacturing. The membranes provide the basis for the fabrication of highly integrated 3D microfluidic devices with a footprint of only 1 × 1 cm(2). When placed on a solid support, the device allows for the immobilization of up to 96 different probes in the form of a 10 × 10 array comprising isolated spots of 50 × 50 µm(2). The design of the channel network is optimized using 3D simulations based on the Lattice-Boltzmann method to promote capillary action as the sole force distributing the liquid in the device. Finally, we demonstrate the patterning of DNA and protein arrays on hard thermoplastic substrates yielding spots of excellent definition that prove to be highly specific in subsequent hybridization experiments.