Andreas Manz

Present state of microchip electrophoresis: state of the art and routine applications.

Microchip electrophoresis (MCE) was one of the earliest applications of the micro-total analysis system (μ-TAS) concept, whose aim is to reduce analysis time and reagent and sample consumption while increasing throughput and portability by miniaturizing analytical laboratory procedures onto a microfluidic chip. More than two decades on, electrophoresis remains the most common separation technique used in microfluidic applications. MCE-based instruments have had some commercial success and have found application in many disciplines. This review will consider the present state of MCE including recent advances in technology and both novel and routine applications in the laboratory. We will also attempt to assess the impact of MCE in the scientific community and its prospects for the future.

A facile in situ microfluidic method for creating multivalent surfaces: toward functional glycomics

An in situ method of modifying the chemistry and topology of microfluidic surfaces in order to mimic the cellular environment is described. The binding of functionalised microbeads to microfluidic channels allows the surface-to-volume ratio of the system, and thus the number of biomolecules available for reaction, to be vastly increased, thereby enhancing the sensitivity of biochemical analyses. The sensitivity and specificity of the technique were first investigated via the study of carbohydrate-protein interactions. Beads featuring hydrazide moieties were adhered to the channel surface, after which carbohydrates (galactose and mannose) were bound to the beads in situ and reacted with fluorescently labelled proteins. Results showed a six-fold increase in fluorescent signal compared to the same process performed on a glass surface without the presence of beads, thereby demonstrating the increase in valence afforded by the method. In a subsequent study, beads, modified with galactose moieties via the in situ functionalisation technique, were used to perform studies of colon tumour cells from a cell sample. Here, the carcinoma cells exhibited superior adhesion than the normal cells due to an increased expression of active galactose receptors, thereby demonstrating the success of the biofunctionalisation method for investigating cellular mechanisms.

On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro

The in vitro study of liver functions and liver cell specific responses to external stimuli deals with the problem to preserve the in vivo functions of primary hepatocytes. In this study, we used the biochip OrganoPlate(TM) (MIMETAS) that combines different advantages for the cultivation of hepatocytes in vitro: (1) the perfusion flow is achieved without a pump allowing easy handling and placement in the incubator; (2) the phaseguides allow plating of matrix-embedded cells in lanes adjacent to the perfusion flow without physical barrier; and (3) the matrix-embedding ensures indirect contact of the cells to the flow. In order to evaluate the applicability of this biochip for the study of hepatocytes functions, Matrigel(TM)-embedded HepG2 cells were cultured over three weeks in this biochip and compared to a static Matrigel culture (3D) and a monolayer culture (2D). Chip-cultured cells grew in spheroid-like structures and were characterized by the formation of bile canaliculi and a high viability over 14 days. Hepatocyte-specific physiology was achieved as determined by an increase in albumin production. Improved detoxification metabolism was demonstrated by strongly increased cytochrome P450 activity and urea production. Additionally, chip-cultured cells displayed increased sensitivity to acetaminophen. Altogether, the OrganoPlate seems to be a very useful alternative for the cultivation of hepatocytes, as their behavior was strongly improved over 2D and static 3D cultures and the results were largely comparable and partly superior to the previous reports on biochip-cultured hepatocytes. As for the low technical needs, this platform has the appearance of being highly applicable for further studies of hepatocytes responses to external stimuli.

Direct coupling of a free-flow isotachophoresis (FFITP) device with electrospray ionization mass spectrometry (ESI-MS)

We present the online coupling of a free-flow isotachophoresis (FFITP) device to an electrospray ionization mass spectrometer (ESI-MS) for continuous analysis without extensive sample preparation. Free-flow-electrophoresis techniques are used for continuous electrophoretic separations using an electric field applied perpendicular to the buffer and sample flow, with FFITP using a discontinuous electrolyte system to concurrently focus a target analyte and remove interferences. The online coupling of FFITP to ESI-MS decouples the separation and detection timeframe because the electrophoretic separation takes place perpendicular to the flow direction, which can be beneficial for monitoring (bio)chemical changes and/or extensive MS(n) studies. We demonstrated the coupling of FFITP with ESI-MS for simultaneous concentration of target analytes and sample clean-up. Furthermore, we show hydrodynamic control of the fluidic fraction injected into the MS, allowing for fluidically controlled scanning of the ITP window. Future applications of this approach are expected in monitoring biochemical changes and proteomics.

Microdroplet technology: Principles and emerging applications

Preface.- Physics of multiphase microflows and microdroplets.- Microfluidic droplet manipulations and its applications.- Active control of droplet formation process in microfluidics.- Recent advances in electrowetting microdroplet technologies.- Automated droplet microfluidic chips for biochemical assays.- The dropletisation of bio-reactions.- Droplet-based microfluidics as a biomimetic principle: from PCR-based virus diagnotics to a generalized concept for handling of biomolecular information.- Droplet microreactors for materials synthesis.- Single-cell analysis.- Trends and perspectives.

Enabling local deposition and controlled synthesis of Au-nanoparticles using a femtopipette

We demonstrate local synthesis and deposition of gold nanoparticles (AuNPs) using a femtopipette, which is a device similar to an atomic force microscope (AFM) sensor but featuring integrated fluidic channels. Two femtopipettes were used, one filled with chloroauric-acid (HAuCl4), and the second with sodium-azide (NaN3). We dispensed two equal droplets (HAuCl4 and NaN3) adjacent to each other, allowing them to coalesce and mix. In this manner, cross-contamination between the tips was prevented. The deposited volumes (per reagent) were 2.4fL, 1.2fL, 0.5fL and 0.25fL. Upon mixture, a chemical reduction process was triggered, which led to the local formation of AuNPs. The performed analysis indicate that the size of the nanoparticles is dominated by droplet volume. For the biggest droplet-mixture, the dominant particle size was 14nm. Whereas for the smallest droplets, the dominant particle size was 4nm. Similarly, the size distribution decreased with smaller droplet volumes. To the best of our knowledge, the combination of deposition and local chemistry using femtoliter-droplets, has not been reported before and represents a new level of compartmentalized nanoparticle synthesis and positioning.

Biocompatibility assay of cellular behavior inside a leaf-inspired biomimetic microdevice at the single-cell level

Herein, we introduce a practical and effective manufacturing methodology for a biomimetic microdevice replicated from the Tilia platyphyllos leaf. With this method, artificial microchambers (of controllable dimension and depth) can be easily integrated into leaf-inspired whole-ordered venation patterns. To display the biocompatibility of this microdevice, we applied it to a long-term (seven days) cell culture and monitored the results. Based on a comprehensive biophysical analysis, including covering cellular deformation, cell migration, cytomembrane tension, extracellular communication, protonema formation, microvilli, and the tethers dynamic of human melanoma cells inside the device at a single-cell resolution, we were able to verify for the first time a leaf-inspired PDMS microdevice as a biocompatible platform for mammal cell culture, showing promise that such a biomimetic device could be further applied for organ-on-a-chip studies and other biomedical research.

Selective and vertical microfabrication of lipid tubule arrays on glass substrates using template-guided gentle hydration

Generally, asymmetric tubular lipid structures have been formed under the specific condition of gentle hydration or by using hydrodynamic and/or electrical elongation of vesicular lipid structures. Small-size lipid tubes are, however, very difficult to allocate or align in the vertical direction on the specific site of the substrate and, therefore, the ability to produce them selectively and in large quantities as an array form is limited. Herein, we propose an easy and novel method to fabricate selective and vertical lipid tube arrays using template-guided gentle hydration of dried lipid films without any external forces. A lipid solution was drop-dispensed onto a porous membrane and dried to form a lipid film. Then, the lipid-coated porous membrane was transferred to a glass substrate by using a UV-cured polymer layer to achieve tight bonding. Upon swelling with an appropriate buffer, expansion forces due to osmotic pressure during the gentle hydration process were highly constrained to confined pores, thereby resulting in the nucleation of tube-like lipid structures through the pores. Interestingly, according to the aspect ratio of pores (ARpore, pore length/pore diameter), different shapes of lipid structures, including vesicular, oval, and tube-like, were generated, which indicates the importance of the ARpore, as well as the pore diameter, during fabrication of tubular lipid structures. Also, this approach was easily modified with 1% chitosan to enhance the stability of the lipid tubes (>30 min in life time), by lipid coating twice and by using unsaturated lipids to increase tube length (>30 μm in length). Therefore, in the future, the simple but robust template-guided gentle hydration method will be a useful tool for fabricating addressable and engineered lipid tube arrays as a sensory unit.

Trends and Perspectives

Throughout the book chapters, researchers have highlighted the recent advancement in microfluidic areas, particularly those involving microdroplets.