Other than concentrating the target molecules at the sensor location, we demonstrate two distinct new advantages of an open-flow impedance-sensing platform for DNA hybridization on carbon nanotube (CNT) surface in the presence of a high-frequency AC electric field. The shear-enhanced DNA and ion transport rate to the CNT surface decouples the parasitic double-layer AC impedance signal from the charge-transfer signal due to DNA hybridization. The flow field at high AC frequency also amplifies the charge-transfer rate across the hybridized CNT and provides shear-enhanced discrimination between DNA from targeted species and a closely related congeneric species with three nucleotide mismatches out of 26 bases in a targeted attachment region. This allows sensitive detection of hybridization events in less than 20 min with picomolar target DNA concentrations in a label-free CNT-based microfluidic detection platform.

Cell spheroids are multicellular aggregates, grown in vitro, that mimic the three-dimensional morphology of physiological tissues. Although there are numerous benefits to using spheroids in cell-based assays, the adoption of spheroids in routine biomedical research has been limited, in part, by the tedious workflow associated with spheroid formation and analysis. Here we describe a digital microfluidic platform that has been developed to automate liquid-handling protocols for the formation, maintenance, and analysis of multicellular spheroids in hanging drop culture. We show that droplets of liquid can be added to and extracted from through-holes, or "wells," and fabricated in the bottom plate of a digital microfluidic device, enabling the formation and assaying of hanging drops. Using this digital microfluidic platform, spheroids of mouse mesenchymal stem cells were formed and maintained in situ for 72 h, exhibiting good viability (>90%) and size uniformity (% coefficient of variation <10% intraexperiment, <20% interexperiment). A proof-of-principle drug screen was performed on human colorectal adenocarcinoma spheroids to demonstrate the ability to recapitulate physiologically relevant phenomena such as insulin-induced drug resistance. With automatable and flexible liquid handling, and a wide range of in situ sample preparation and analysis capabilities, the digital microfluidic platform provides a viable tool for automating cell spheroid culture and analysis.

Cell invasion is a key process in tissue growth, wound healing, and tumor progression. Most invasion assays examine cells cultured in adherent monolayers, which fail to recapitulate the three-dimensional nuances of the tissue microenvironment. Multicellular cell spheroids have a three-dimensional (3D) morphology and mimic the intercellular interactions found in tissues in vivo, thus providing a more physiologically relevant model for studying the tissue microenvironment and processes such as cell invasion. Spheroid-based invasion assays often require tedious, manually intensive handling protocols or the use of robotic liquid handling systems, which can be expensive to acquire, operate, and maintain. Here we describe a digital microfluidic (DμF) platform that enables formation of spheroids by the hanging drop method, encapsulation of the spheroids in collagen, and the exposure of spheroids to migration-modulating agents. Collagen sol-gel solutions up to 4 mg mL(-1), which form gels with elastic moduli up to ∼50 kPa, can be manipulated on the device. In situ spheroid migration assays show that cells from human fibroblast spheroids exhibit invasion into collagen gels, which can be either enhanced or inhibited by the delivery of exogenous migration modulating agents. Exposing fibroblast spheroids to spheroid secretions from colon cancer spheroids resulted in a >100% increase in fibroblast invasion into the collagen gel, consistent with the cancer-associated fibroblast phenotype. These data show that DμF can be used to automate the liquid handling protocols for spheroid-based invasion assays and create a cell invasion model that mimics the tissue microenvironment more closely than two-dimensional culturing techniques do. A DμF platform that facilitates the creation and assaying of 3D in vitro tissue models has the potential to make automated 3D cell-based assays more accessible to researchers in the life sciences.