Coupling Miniaturized Free-Flow Electrophoresis to Mass Spectrometry via a Multi-Emitter ESI Interface.

We present a novel multi-emitter electrospray ionization (ESI) interface for the coupling of microfluidic free-flow electrophoresis (µFFE) with mass spectrometry (MS). The effluents of the µFFE outlets are analyzed in near real-time, allowing a direct optimization of the electrophoretic separation and an online monitoring of qualitative sample compositions. The short measurement time of just a few seconds for all outlets even enables a reasonable time-dependent monitoring. As a proof of concept, we employ the multi-emitter ESI interface for the continuous identification of analytes at 15 µFFE outlets via MS to optimize the µFFE separation of important players of cellular respiration in operando. The results indicate great potential of the presented system in downstream processing control, for example, for the monitoring and purification of products in continuous-flow microreactors.

Multiplexed Online Monitoring of Microfluidic Free-Flow Electrophoresis via Mass Spectrometry.

Free-flow electrophoresis is a tool for the continuous fractionation of electrically charged analytes. In this study, we introduce a novel method to couple microchip-based free-flow electrophoresis with mass spectrometry. The successive connection of multiple microchip outlets to the electrospray ionization source of a mass spectrometer is automated using a multiposition valve. With this novel setup, it is possible to continuously fractionate and collect compounds while simultaneously monitoring the process online with mass spectrometry. The functionality of the method is demonstrated by the successful separation and identification of the biomolecules AMP, ATP, and CoA, which are fundamental for numerous biochemical processes in every organism.

Flow rate independent gradient generator and application in microfluidic free-flow electrophoresis.

Microfluidic gradient generators have been employed in several works in the literature. However, these are typically application specific and especially limited in the range of flow rates that result in the required concentration gradient outputs. Here, a flow rate independent gradient generator designed as a modified Christmas tree-like microfluidic channel network including micromixers at each channel branch is demonstrated. The device was characterized theoretically, modeled using finite element analysis and tested experimentally. Input flow rates up to 200 µl/min, resulting in a maximum speed of about 333 mm/s, for the generation of linear and mirrored linear gradients were demonstrated. As an application example, the gradient generator was monolithically integrated with microfluidic free-flow electrophoresis for the separation/concentration of fluorophores using a novel E-field gradient free-flow electrophoresis mode. The separation of fluorophores, having different charge stages, showed concentration factors of up to 10 fold. In addition, an extended theoretical description of the realizable concentration gradients and the electric field gradient is presented as supplementary information.

Nanoliter Sensing for Infrared Bioanalytics.

Nondestructive label-free bioanalytics of microliter to nanoliter sample volumes with low analyte concentrations requires novel analytic approaches. For this purpose, we present an optofluidic platform that combines surface-enhanced in situ infrared spectroscopy with microfluidics for sensing of surface-immobilized ultrathin biomolecular films in liquid analytes. Submonolayer sensitivity down to surface densities of few ng/cm2 is demonstrated for the adsorption of the thiolate tripeptide glutathione and for the recognition of streptavidin on a biotinylated enhancement substrate. Nonfunctionalized and functionalized metal island films on planar oxidized silicon substrates are used for signal enhancement with quantifiable enhancement properties. A single-reflection geometry at an incidence angle below the attenuated-total-reflection (ATR) regime is used with ordinary planar, IR-transparent windows. The geometry circumvents the strong IR absorption of common polymer materials and of aqueous environments in the IR fingerprint region. This practice enables straightforward quantitative analyses of, e.g., adsorption kinetics as well as chemical and structural properties in dependence of external stimuli.

Integration of polycarbonate membranes in microfluidic free-flow electrophoresis.

A general difficulty in the miniaturization of free-flow electrophoresis relates to the need to separate electrodes and separation bed compartments. This is usually performed by using membranes, which are either difficult to fabricate and integrate into microfluidic channels, or not stable over time. Here, we propose the use of track-etched polycarbonate membranes. Fabrication of the miniaturized device and integration of the membrane was simple, reproducible and allows for long shelf times. Furthermore, the membranes were resistant to high pressure values (up to 105 Pa), and contributed negligible electrical resistance, allowing setting of electric fields at the separation bed with high efficiency. A second microfluidic device was connected to the microfluidic free-flow electrophoresis chip via tubing, ensured flow stability over time and was used as a chip-to-world interface to a 96 well plate. We demonstrated microfluidic free-flow zone- and field-stacking electrophoresis, and isoelectric focusing proof-of-principle experiments, using fluorescent analytes and monitoring via fluorescence microscopy. Furthermore, the separation of a mixture of 7 proteins was performed in microfluidic free-flow zone electrophoresis mode. Subsequent analysis via protein mass spectrometry of the collected fractions revealed separation of the protein mixture, indicating a wide range of applications in the characterization of proteins and biosimilars.

Current advances and challenges in microfluidic free-flow electrophoresis-A critical review.

The research field on microfluidic free-flow electrophoresis has developed vast amounts of devices, methods, applications and raised new questions, often in analogy to conventional techniques from which it derives. Most efforts have been employed on device development and a myriad of architectures and fabrication techniques have been reported using simple proof-of-principle separations. As technological aspects reach a quite mature state, researchers' new challenges include the development of protocols for the separation of complex mixtures, as required in the fields of application. The success of this effort is extremely dependent on the capability to transfer the device's fabrication to an industrial setting as well as to ensure interfacing simplicity, namely at the solutions' supply and collection, and actuation such as electric potential application and temperature control. Other advanced applications such as direct interfacing to downstream systems such as mass spectrometry, integration of sensing and feedback controls will require further development in the laboratory. In this review we provide an overview on the field, from basic concepts, through advanced developments both in the theoretical and experimental arenas, and addressing the above details. A comprehensive survey of designs, materials and applications is presented with particular highlights to most recent developments, namely the integration of electrodes, flow control and hyphenation of microfluidic free-flow electrophoresis with other techniques.

Correction: Micropatterning neuronal networks.

Correction for 'Micropatterning neuronal networks' by Heike Hardelauf, et al., Analyst, 2014, 139, 3256-3264.

High spatial and temporal resolution cell manipulation techniques in microchannels.

The advent of microfluidics has enabled thorough control of cell manipulation experiments in so called lab on chips. Lab on chips foster the integration of actuation and detection systems, and require minute sample and reagent amounts. Typically employed microfluidic structures have similar dimensions as cells, enabling precise spatial and temporal control of individual cells and their local environments. Several strategies for high spatio-temporal control of cells in microfluidics have been reported in recent years, namely methods relying on careful design of the microfluidic structures (e.g. pinched flow), by integration of actuators (e.g. electrodes or magnets for dielectro-, acousto- and magneto-phoresis), or integrations thereof. This review presents the recent developments of cell experiments in microfluidics divided into two parts: an introduction to spatial control of cells in microchannels followed by special emphasis in the high temporal control of cell-stimulus reaction and quenching. In the end, the present state of the art is discussed in line with future perspectives and challenges for translating these devices into routine applications.

Emitter-assigned multi-dielectric barrier-nano-electrospray ionization mass spectrometry.

A multi-dielectric-barrier-nano-electrospray ionization (multi-DB-nESI) emitter setup is presented where the emitters are quasi-simultaneously switched to ignite the respective spray solving the problem of coulombic interferences. Since the switching is done electronically, the sprays can be synchronized to the mass spectrometry (MS) ion trap and the resulting mass spectra can be assigned to the corresponding nESI emitter. Graphical Abstract Multi-dielectric-barrier-nano-electrospray in front of a mass spectrometer inlet.

Preparation of neuronal co-cultures with single cell precision.

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (<10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (>85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Micropatterning neuronal networks.

Spatially organised neuronal networks have wide reaching applications, including fundamental research, toxicology testing, pharmaceutical screening and the realisation of neuronal implant interfaces. Despite the large number of methods catalogued in the literature there remains the need to identify a method that delivers high pattern compliance, long-term stability and is widely accessible to neuroscientists. In this comparative study, aminated (polylysine/polyornithine and aminosilanes) and cytophobic (poly(ethylene glycol) (PEG) and methylated) material contrasts were evaluated. Backfilling plasma stencilled PEGylated substrates with polylysine does not produce good material contrasts, whereas polylysine patterned on methylated substrates becomes mobilised by agents in the cell culture media which results in rapid pattern decay. Aminosilanes, polylysine substitutes, are prone to hydrolysis and the chemistries prove challenging to master. Instead, the stable coupling between polylysine and PLL-g-PEG can be exploited: Microcontact printing polylysine onto a PLL-g-PEG coated glass substrate provides a simple means to produce microstructured networks of primary neurons that have superior pattern compliance during long term (>1 month) culture.

Whole cell quenched flow analysis.

This paper describes a microfluidic quenched flow platform for the investigation of ligand-mediated cell surface processes with unprecedented temporal resolution. A roll-slip behavior caused by cell-wall-fluid coupling was documented and acts to minimize the compression and shear stresses experienced by the cell. This feature enables high-velocity (100-400 mm/s) operation without impacting the integrity of the cell membrane. In addition, rotation generates localized convection paths. This cell-driven micromixing effect causes the cell to become rapidly enveloped with ligands to saturate the surface receptors. High-speed imaging of the transport of a Janus particle and fictitious domain numerical simulations were used to predict millisecond-scale biochemical switching times. Dispersion in the incubation channel was characterized by microparticle image velocimetry and minimized by using a horizontal Hele-Shaw velocity profile in combination with vertical hydrodynamic focusing to achieve highly reproducible incubation times (CV = 3.6%). Microfluidic quenched flow was used to investigate the pY1131 autophosphorylation transition in the type I insulin-like growth factor receptor (IGF-1R). This predimerized receptor undergoes autophosphorylation within 100 ms of stimulation. Beyond this demonstration, the extreme temporal resolution can be used to gain new insights into the mechanisms underpinning a tremendous variety of important cell surface events.

Dielectric barrier electrospray-polarity cycle and trigger.

The parameters influencing the combination of the dielectric barrier electrospray (DB-ES) with an ion trap mass spectrometer are investigated. Two approaches are presented: the application of different polarity cycles in the DB electrospray high voltage signal and the triggering of it to an output signal received by the mass spectrometer. Both approaches are addressed to improve the detection sensitivity over the sensitivity of conventional nano ES.

Microfluidic construction of minimalistic neuronal co-cultures.

In this paper we present compartmentalized neuron arraying (CNA) microfluidic circuits for the preparation of neuronal networks using minimal cellular inputs (10-100-fold less than existing systems). The approach combines the benefits of microfluidics for precision single cell handling with biomaterial patterning for the long term maintenance of neuronal arrangements. A differential flow principle was used for cell metering and loading along linear arrays. An innovative water masking technique was developed for the inclusion of aligned biomaterial patterns within the microfluidic environment. For patterning primary neurons the technique involved the use of meniscus-pinning micropillars to align a water mask for plasma stencilling a poly-amine coating. The approach was extended for patterning the human SH-SY5Y neuroblastoma cell line using a poly(ethylene glycol) (PEG) back-fill and for dopaminergic LUHMES neuronal precursors by the further addition of a fibronectin coating. The patterning efficiency Epatt was >75% during lengthy in chip culture, with ∼85% of the outgrowth channels occupied by neurites. Neurons were also cultured in next generation circuits which enable neurite guidance into all outgrowth channels for the formation of extensive inter-compartment networks. Fluidic isolation protocols were developed for the rapid and sustained treatment of the different cellular and sub-cellular compartments. In summary, this research demonstrates widely applicable microfluidic methods for the construction of compartmentalized brain models with single cell precision. These minimalistic ex vivo tissue constructs pave the way for high throughput experimentation to gain deeper insights into pathological processes such as Alzheimer and Parkinson Diseases, as well as neuronal development and function in health.

Measured effects of various electrolyte and capillary properties in dielectric barrier electrospray ionization: development of a comprehensive model.

The mechanism which leads to a dielectric barrier electrospray (DB-ES) was investigated by measurements of the electrical current. It comprises several components, namely, the displacement current, currents which are initiated by electromagnetic transmission, an ion current inside the capillary, which is responsible for the built-up of the potential at the tip, and the electrospray current as a result of the displacement current and the ion transfer current. An augmented interpretation of the current signal of the DB-ES is presented, and features are described in more detail. The behavior of the ions inside the capillary and the charge transfer are described from the chemical and the electrical point of view.

A microfluidic array with cellular valving for single cell co-culture.

We present a highly parallel microfluidic approach for contacting single cell pairs. The approach combines a differential fluidic resistance trapping method with a novel cellular valving principle for homotypic and heterotypic single cell co-culturing. Differential fluidic resistance was used for sequential single cell arraying, with the adhesion and flattening of viable cells within the microstructured environment acting to produce valves in the open state. Reversal of the flow was used for the sequential single cell arraying of the second cell type. Plasma stencilling, along the linear path of least resistance, was required to confine the cells within the trap regions. Prime flow conditions with minimal shear stress were identified for highly efficient cell arraying (∼99%) and long term cell culture. Larger trap dimensions enabled the highest levels of cell pairing (∼70%). The single cell co-cultures were in close proximity for the formation of connexon structures and the study of contact modes of communication. The research further highlights the possibility of using the natural behaviour of cells as the working principle behind responsive microfluidic elements.

Characterization of dielectric barrier electrospray ionization for mass spectrometric detection.

A novel electrospray interface is presented which induces an electric field by dielectric polarization through a non-conductive barrier. Therefore, a square-wave high-voltage signal is applied. This technique allows mass spectrometric measurements in the positive as well as in the negative mass spectrometry mode without changing the polarity of the potential applied, and it decreases the risk of undesired discharges, induced by high electric currents. The applicability of this technique is demonstrated by mass spectrometric determination of reserpine.

Temperature gradient focusing in miniaturized free-flow electrophoresis devices.

Temperature gradient focusing is a method to separate and focus any charged analytes even without accessible isoelectric point, and has been already widely used in CE. In this paper, we demonstrate the application of temperature gradient focusing to free-flow electrophoresis. Besides focusing and separation experiments of proteins, the stability of the temperature gradient under flow conditions and the temperature dependence of fluorescence dyes have also been investigated.

Separation of proteins using a novel two-depth miniaturized free-flow electrophoresis device with multiple outlet fractionation channels.

A novel free-flow electrophoresis glass chip design with two-depth etched structures for the separation and fractionation of proteins is presented. The microfluidic structures etched in two depths enhance the flow characteristics inside the miniaturized device. A novel nine-port outlet interface enables the fractionation of the separated analytes. The separation and focussing of a protein sample mixture demonstrated the ability of the new chip.

Isotachophoretic free-flow electrophoretic focusing and SERS detection of myoglobin inside a miniaturized device.

A new approach for label-free optical sample detection via surface enhanced Raman spectroscopy (SERS) inside a free-flow electrophoresis (FFE) chip is presented in this work.