Melanoma cells can switch between a melanocytic and mesenchymal-like state. Scattered evidence indicates that additional, intermediate state(s) may exist. To search for such states and decipher their underlying gene regulatory network (GRN), we studied ten melanoma cultures by single-cell RNA-seq, and 26 additional cultures by bulk RNA-seq. Although each culture exhibited a unique transcriptome, we identified shared GRNs that underlie the extreme melanocytic and mesenchymal states, and the intermediate state. This intermediate state is corroborated by a distinct chromatin landscape and governed by the transcription factors SOX6, NFATC2, EGR3, ELF1 and ETV4. Single-cell migration assays confirmed its intermediate migratory phenotype. By time-series sampling of single cells after knockdown of SOX10, we unravelled the sequential and recurrent arrangement of GRNs during phenotype switching. Jointly, these analyses indicate that an intermediate state exists and is driven by a distinct and stable "mixed" GRN rather than being a symbiotic, heterogeneous mix of cells.

Digital microfluidics is introduced as a novel platform with unique advantages for performing single-molecule detection. We demonstrate how superparamagnetic beads, used for capturing single protein molecules, can be printed with unprecedentedly high loading efficiency and single bead resolution on an electrowetting-on-dielectric-based digital microfluidic chip by micropatterning the Teflon-AF surface of the device. By transporting droplets containing suspended superparamagnetic beads over a hydrophilic-in-hydrophobic micropatterned Teflon-AF surface, single beads are trapped inside the hydrophilic microwells due to their selective wettability and tailored dimensions. Digital microfluidics presents the following advantages for printing and sealing magnetic beads for single-molecule detection: (i) droplets containing suspended beads can be transported back and forth over the array of hydrophilic microwells to obtain high loading efficiencies of microwells with single beads, (ii) the use of hydrophilic-in-hydrophobic patterns permits the use of a magnet to speed up the bead transfer process to the wells, while the receding droplet meniscus removes excess beads off the chip surface and thereby shortens the bead patterning time, and (iii) reagents can be transported over the printed beads multiple times, while capillary forces and a magnet hold the printed beads in place. High loading efficiencies (98% with a CV of 0.9%) of single beads in microwells were obtained by transporting droplets of suspended beads over the array 10 times in less than 1 min, which is much higher than previously reported methods (40-60%), while the total surface area needed for performing single-molecule detection can be decreased. The performance of the device was demonstrated by fluorescent detection of the presence of the biotinylated enzyme β-galactosidase on streptavidin-coated beads with a linear dynamic range of 4 orders of magnitude ranging from 10 aM to 90 fM.

Electrowetting-on-dielectric (EWOD) lab-on-a-chip systems have already proven their potential within a broad range of bio-assays. Nevertheless, research on the analytical performance of those systems is limited, yet crucial for a further breakthrough in the diagnostic field. Therefore, this paper presents the intrinsic possibilities of an EWOD lab-on-a-chip as a versatile platform for homogeneous and heterogeneous bio-assays with high analytical performance. Both droplet dispensing and splitting cause variations in droplet size, thereby directly influencing the assay's performance. The extent to which they influence the performance is assessed by a theoretical sensitivity analysis, which allows the definition of a basic framework for the reduction of droplet size variability. Taking advantage of the optimized droplet manipulations, both homogeneous and heterogeneous bio-assays are implemented in the EWOD lab-on-a-chip to demonstrate the analytical capabilities and versatility of the device. A fully on-chip enzymatic assay is realized with high analytical performance. It demonstrates the promising capabilities of an EWOD lab-on-a-chip in food-related and medical applications, such as nutritional and blood analyses. Further, a magnetic bio-assay for IgE detection using superparamagnetic nanoparticles is presented whereby the nanoparticles are used as solid carriers during the bio-assay. Crucial elements are the precise manipulation of the superparamagnetic nanoparticles with respect to dispensing and separation. Although the principle of using nano-carriers is demonstrated for protein detection, it can be easily extended to a broader range of bio-related applications like DNA sensing. In heterogeneous bio-assays the chip surface is actively involved during the execution of the bio-assay. Through immobilization of specific biological compounds like DNA, proteins and cells a reactive chip surface is realized, which enhances the bio-assay performance. To demonstrate this potential, on-chip adhesion islands are fabricated to immobilize MCF-7 human breast cancer cells. Viability studies are performed to assess the functionalization efficiency.

In this paper we report on the controlled biofunctionalization of the hydrophobic layer of electrowetting-on-dielectric (EWOD) based microfluidic chips with the aim to execute (adherent) cell-based assays. The biofunctionalization technique involves a dry lift-off method with an easy to remove Parylene-C mask and allows the creation of spatially controlled micropatches of biomolecules in the Teflon-AF(®) layer of the chip. Compared to conventional methods, this method (i) is fully biocompatible; and (ii) leaves the hydrophobicity of the chip surface unaffected by the fabrication process, which is a crucial feature for digital microfluidic chips. In addition, full control of the geometry and the dimensions of the micropatches is achieved, allowing cells to be arrayed as cell clusters or as single cells on the digital microfluidic chip surface. The dry Parylene-C lift-off technique proves to have great potential for precise biofunctionalization of digital microfluidic chips, and can enhance their use for heterogeneous bio-assays that are of interest in various biomedical applications.

Protein drugs, e.g. hormonal drugs, cannot be delivered orally to a patient as they get digested in the gastro-intestinal (GI) tract. Thus, it is imperative that these kinds of drugs are delivered transdermally through the skin. To provide for real-time feedback as well as to test independently for various substances in the blood, we also need a blood sampling system. Microneedles can perform both these functions. Further, microneedles made of silicon or metal have the risk of breaking inside the skin thereby leading to complications. SU-8, being approved of as being biocompatible by the Food and Drug Agency (FDA) of the United States, is an attractive alternative because firstly it is a polymer material, thereby reducing the chances of breakages inside the skin, and secondly it is a negative photoresist, thereby leading to ease of fabrication. Thus, here we present very tall (around 1600 μm) SU-8 polymer-based hollow microneedles fabricated by a simple and repeatable process, which are a very good candidate for transdermal drug delivery as well as blood extraction. The paper elaborates on the details that allow the fabrication of such extreme aspect ratios (>100).