Control and automation of multilayered integrated microfluidic device fabrication.

Integrated microfluidics is a sophisticated three-dimensional (multi layer) solution for high complexity serial or parallel processes. Fabrication of integrated microfluidic devices requires soft lithography and the stacking of thin-patterned PDMS layers. Precise layer alignment and bonding is crucial. There are no previously reported standards for alignment of the layers, which is mostly performed using uncontrolled processes with very low alignment success. As a result, integrated microfluidics is mostly used in academia rather than in the many potential industrial applications. We have designed and manufactured a semiautomatic Microfluidic Device Assembly System (µDAS) for full device production. µDAS comprises an electrooptic mechanical system consisting of four main parts: optical system, smart media holder (for PDMS), a micropositioning xyzθ system and a macropositioning XY mechanism. The use of the µDAS yielded valuable information regarding PDMS as the material for device fabrication, revealed previously unidentified errors, and enabled optimization of a robust fabrication process. In addition, we have demonstrated the utilization of the µDAS technology for fabrication of a complex 3 layered device with over 12 000 micromechanical valves and an array of 64 × 64 DNA spots on a glass substrate with high yield and high accuracy. We increased fabrication yield from 25% to about 85% with an average layer alignment error of just ∼4 µm. It also increased our protein expression yields from 80% to over 90%, allowing us to investigate more proteins per experiment. The µDAS has great potential to become a valuable tool for both advancing integrated microfluidics in academia and producing and applying microfluidic devices in the industry.

Screening for Host Factors Directly Interacting with RSV Protein: Microfluidics.

We present a high-throughput microfluidics platform to identify novel host cell binding partners of respiratory syncytial virus (RSV) matrix (M) protein. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed custom-made gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for binding to RSV M protein.Even small viral proteome, such as that of RSV, presents a challenge due to the fact that viral proteins are usually multifunctional and thus their interaction with the host is complex. Protein microarrays technology allows the interrogation of protein-protein interactions, which could possibly overcome obstacles by using conventional high throughput methods. Using microfluidics platform we have identified new host interactors of M involved in various cellular pathways. A number of microfluidics based assays have already provided novel insights into the virus-host interactome, and the results have important implications for future antiviral strategies aimed at targets of viral protein interactions with the host.

New host factors important for respiratory syncytial virus (RSV) replication revealed by a novel microfluidics screen for interactors of matrix (M) protein.

Although human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and elderly worldwide, there is no licensed RSV vaccine or effective drug treatment available. The RSV Matrix protein plays key roles in virus life cycle, being found in the nucleus early in infection in a transcriptional inhibitory role, and later localizing in viral inclusion bodies before coordinating viral assembly and budding at the plasma membrane. In this study, we used a novel, high throughput microfluidics platform and custom human open reading frame library to identify novel host cell binding partners of RSV matrix. Novel interactors identified included proteins involved in host transcription regulation, the innate immunity response, cytoskeletal regulation, membrane remodeling, and cellular trafficking. A number of these interactions were confirmed by immunoprecipitation and cellular colocalization approaches. Importantly, the physiological significance of matrix interaction with the actin-binding protein cofilin 1, caveolae protein Caveolin 2, and the zinc finger protein ZNF502 was confirmed. siRNA knockdown of the host protein levels resulted in reduced RSV virus production in infected cells. These results have important implications for future antiviral strategies aimed at targets of RSV matrix in the host cell.

The nucleolar PICT-1/GLTSCR2 protein forms homo-oligomers.

The human "protein interacting with carboxyl terminus 1" (PICT-1), also designated as the "glioma tumor suppressor candidate region 2 gene product", GLTSCR2, is a nucleolar protein whose activity is, as yet, unknown. Contradictory results regarding the role of PICT-1 in cancer have been reported, and PICT-1 has been suggested to function either as a tumor suppressor protein or as an oncogene. In this study, we demonstrate self-association of PICT-1. Through yeast two-hybrid assay, we identified PICT-1 as its own interaction partner. We confirmed the interaction of PICT-1 with itself by direct yeast two-hybrid assay and also showed self-association of PICT-1 in mammalian cells by co-immunoprecipitation and fluorescence resonance energy transfer assays. Furthermore, we confirmed direct self-association of PICT-1 by using in vitro microfluidic affinity binding assays. The later assay also identified the carboxy-terminal domain as mediating self-interaction of PICT-1. Glutaraldehyde cross-linking and gel-filtration assays suggest that PICT-1 forms dimers, though it may form higher-order complexes as well. Our findings add another layer of complexity in understanding the different functions of PICT-1 and may help provide insights regarding the activities of this protein.

Microfluidic large scale integration of viral-host interaction analysis.

Viral-host interactions represent potential drug targets for novel antiviral strategies (Flisiak et al., Hepatology, 2008, 47, 817-26). Hence, it is important to establish an adequate platform for identifying and analyzing such interactions. In this review, we discuss bottlenecks in conventional protein-protein interaction methodologies and present the contribution of innovative microfluidic-based technologies towards a solution to these problems with respect to viral-host proteomics.