Klaus Eyer

Interfacing droplet microfluidics with matrix-assisted laser desorption/ionization mass spectrometry: label-free content analysis of single droplets.

Droplet-based microfluidic systems have become a very powerful tool to miniaturize chemical and biological reactions. However, droplet content analysis remains challenging and relies almost exclusively on optical methods such as fluorescence spectroscopy. Hence, labeling of the analyte is typically required which impedes a more universal applicability of microdroplets. Here we present a novel interface coupling droplet microfluidics and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for label-free content analysis of single droplets. Nanoliter aqueous droplets immersed in perfluorinated oil are created in a microfluidic T-junction, transferred into a capillary, and deposited on a high-density microarray MALDI plate mounted on a motorized xy-stage. The fully automated system is robust and reliable due to two unique features. First, a simple optical droplet detection system is used to synchronize stage movement and exit of droplets from the capillary. Second, the microarray plate contains an array of over 26,000 hydrophilic spots within a hydrophobic coating, each spot acting as a recipient to confine the droplets and to prevent cross-contamination. The MALDI matrix can also be applied using our system by spotting matrix droplets on the microarray in a separate run. To demonstrate the potential of our system, we studied the enzymatic cleavage of angiotensin I by angiotensin converting enzyme and monitored the increasing concentration of the product angiotensin II over time. The interface provides a robust and fully automated method for rapid label-free and information-rich content analysis of single droplets. With the high number of droplets per plate, this method is particularly suitable for high-throughput screening applications.

Implementing Enzyme-Linked Immunosorbent Assays on a Microfluidic Chip To Quantify Intracellular Molecules in Single Cells

Cell-to-cell differences play a key role in the ability of cell populations to adapt and evolve, and they are considered to impact the development of several diseases. Recent advances in microsystem technology provide promising solutions for single-cell studies. However, the quantitative chemical analysis of single-cell lysates remains difficult. Here, we combine a microfluidic device with the analytical strength of enzyme-linked immunosorbent assays (ELISA) for single-cell studies to reliably identify intracellular proteins, secondary messengers, or metabolites. The microfluidic device allows parallel single-cell trapping and isolation in 625-pL microchambers, repeated treatment and washing steps, subsequent lysis and analysis by ELISA. Using a sandwich ELISA, we quantitatively determined the concentration of the enzyme GAPDH in single U937 cells and HEK 293 cells, and found amounts within a range of a few (1-4) attomol per cell. Furthermore, a competitive ELISA is performed to determine the concentration of the secondary messenger cyclic adenosine monophosphate (cAMP) in MLT cells, in response to the hormone lutropin. We found the half maximal effective concentration (EC50) of lutropin to have an average value of 2.51 ± 0.44 ng/mL. Surprisingly, there were large cell-to-cell variations for all supplied lutropin concentrations, ranging from 36 to 536 attomol cAMP for nonstimulated cells and from 80 to 1040 attomol cAMP for a concentration around the EC50 (3 ng/mL). Because of the high sensitivity and specificity of ELISA and the large number of antibodies available, we believe that our device provides a new, powerful means for single-cell proteomics and metabolomics.

A new mechanobiological era: microfluidic pathways to apply and sense forces at the cellular level.

Fueled by technological advances in micromanipulation methodologies, the field of mechanobiology has boomed in the last decade. Increasing needs for clinical solutions to better maintain our major mechanosensitive tissues (muscle, bone, and cartilage) with increasing age and new insights into cellular adaptations to mechanical stresses beckon for novel approaches to meet the needs of the future. In particular, the emergence of microfluidics has inspired new interdisciplinary strategies to decipher cellular mechanotransduction on the biochemical as well as macromolecular level. Cellular actuation by locally varying fluid shear can serve to accurately alter membrane surface tension as well as produce direct compressive and strain forces onto cells. Moreover, incorporating microelectronic technologies into microfluidic platforms has led to further advances in actuation and readout possibilities. In this review, we discuss the application of microfluidics to mechanobiological research with particular focus on microfluidic platforms that are able to simultaneously monitor cellular adaptation to mechanical forces and interpret biochemical mechanotransduction.

Microfluidic trapping of giant unilamellar vesicles to study transport through a membrane pore

We present a microfluidic platform able to trap single GUVs in parallel. GUVs are used as model membranes across many fields of biophysics including lipid rafts, membrane fusion, and nanotubes. While their creation is relatively facile, handling and addressing single vesicles remains challenging. The PDMS microchip used herein contains 60 chambers, each with posts able to passively capture single GUVs without compromising their integrity. The design allows for circular valves to be lowered from the channel ceiling to isolate the vesicles from rest of the channel network. GUVs containing calcein were trapped and by rapidly opening the valves, the membrane pore protein α-hemolysin (αHL) was introduced to the membrane. Confocal microscopy revealed the kinetics of the small molecule efflux for different protein concentrations. This microfluidic approach greatly improves the number of experiments possible and can be applied to a wide range of biophysical applications.

A microfluidic vesicle screening platform: monitoring the lipid membrane permeability of tetracyclines.

For many drugs including antibiotics such as tetracyclines it is crucial that the molecule has the ability to quickly and passively permeate lipid membranes. Hence, the understanding of the permeability in relation to the molecular structure is an important aspect to rationally design novel pharmaceutically active compounds with high bioavailability. Here, we present a versatile method to study the kinetics of tetracycline permeation across liposome membranes on a microchip. Liposomes are immobilized onto the glass surface in a stripe pattern via an avidin-biotin bond and covered by microchannels to allow continuous delivery of tetracycline and buffer. The fluid flow provides a constant concentration profile and thereby resembles the drug transport via blood in the human body. Total internal reflection fluorescence (TIRF) microscopy was used to image the formation of a fluorescent drug-europium complex inside the liposomes. The permeation rates of various tetracyclines were investigated and the results compared to a conventional method (water-octanol partitioning). The findings largely confirm the correlation between membrane permeability and lipophilicity of the permeating molecules (Overtons rule). However, slight deviations reveal that lipophilicity is an important but not the exclusive parameter for the prediction of permeation. The method is fast enough to study the permeation of unstable tetracyclines such as rolitetracycline. Additionally, with the use of different cholesterol concentrations, the influence of membrane composition on the permeation rate can be investigated conveniently. The microfluidic approach can be easily applied to investigate the kinetics of other processes such as ligand-membrane receptor association and dissociation, provided that the process can be visualized by means of fluorescence spectroscopy.

A facile protocol for the immobilisation of vesicles, virus particles, bacteria, and yeast cells

Immobilisation of liposomes and cells is often a prerequisite for long-term observations. The most common immobilisation approaches rely on surface modifications, encapsulation in porous materials or trapping in microfluidic channels by means of hurdle-like structures. While these approaches are useful for larger mammalian cells, the immobilisation of smaller organisms like bacteria and yeast or membrane model systems such as liposomes typically requires modification of their outer membrane to ensure that they are stably arrested at a defined position. Here, we present a protocol to immobilise biological objects, which can interact with hydrophobic cholesterol. A water-soluble molecule (cholesterol-PEG-biotin) is used as a linker, which can bind via avidin to biotinylated BSA (bBSA) previously absorbed on a glass surface. For better visualization, bBSA is arranged in a dot pattern by means of microcontact printing, and a microfluidic channel is used for sample supply. We show that our approach can be used to successfully immobilise artificial liposomes of different sizes, native (cell-derived) vesicles, vaccinia virions, Saccharomyces cerevisiae and Escherichia coli, simply by flushing the objects through the channel. Under these conditions, small liposomes and biological objects are stably arrested at high flow rates, while larger cells and liposomes can be released again by application of high shear stress. This protocol can be applied for long-term studies where fluids must be changed repeatedly, for measuring fast kinetics where rapid fluid exchange is essential, and to study the effects of shear stress.

On-chip enzyme quantification of single Escherichia coli bacteria by immunoassay-based analysis.

Individual bacteria of an isogenic population can differ significantly in their phenotypic characteristics. This cellular heterogeneity is thought to increase the adaptivity to environmental changes on a population level. Analytical methods for single-bacteria analyses are essential to reveal the different factors that may contribute to this cellular heterogeneity, among them the stochastic gene expression, cell cycle stages and cell aging. Although promising concepts for the analysis of single mammalian cells based on microsystems technology were recently developed, platforms suitable for proteomic analyses of microbial cells are by far more challenging. Here, we present a microfluidic device optimized for the analysis of single Escherichia coli bacteria. Individual bacteria are captured in a trap and isolated in a volume of only 155 pL. In combination with an immunoassay-based analysis of the cell lysate, the platform allowed the selective and sensitive analysis of intracellular enzymes. The limit of detection of the developed protocol was found to be 200 enzymes. Using this platform, we could investigate the levels of β-galactosidase in cells grown under different nutrient conditions. We successfully determined the enzyme copy numbers in cells cultured in defined medium (3517 ± 1578) and in complex medium (4710 ± 2643), and verified the down-regulation of expression in medium that contained only glucose as carbon source. The strong variations we found for individual bacteria confirm the phenotype heterogeneity. The capability to quantify proteins and other molecules in single bacterial lysates is encouraging to use the new analysis platform in future proteomics studies of isogenic bacteria populations.

Controllable electrofusion of lipid vesicles: initiation and analysis of reactions within biomimetic containers

We present a microfluidic device that is able to trap multiple giant unilamellar vesicles (GUVs) and initiate electrofusion via integrated microelectrodes. PDMS posts were designed to trap and isolate two or more vesicles. Electrodes patterned onto the glass surface of the microchannels are able to apply a short, high voltage pulse across the traps for controllable electrofusion of the GUVs. The entire array of traps and electrodes are designed such that an average of 60 individual fusion experiments can be performed on-chip. An assay based on Förster resonance energy transfer (FRET) is performed to show successful lipid mixing. Not only can the device be used to record the dynamics of lipid membrane fusion, but it can be used for reaction monitoring by fusing GUVs containing reactants. We demonstrate this by fusing vesicles encapsulating femtolitre volumes of cobalt chloride or EDTA and monitoring the amount of the complexation product over time.

Enantiomeric and Diastereoisomeric (Mixed) L/ D-Octaarginine Derivatives – A Simple Way of Modulating the Properties of Cell-Penetrating Peptides†

Cell‐penetrating peptides (CPPs) are promising vehicles for delivery of drugs, antibiotics, proteins, nucleic acid derivatives, etc. into eukaryotic and prokaryotic target cells. To prevent premature degradation, CPPs consisting of D‐ or β‐amino acid residues have been used. We present simple models for the various modes of delivery of physiologically active cargoes by CPPs, depending on the nature of their conjugation (Fig. 1), and we describe the plasma stability of oligoarginines (OAs) 1–4, the most common unnatural CPPs. Fluorescein‐labeled L‐octaarginine 1 was found to have a half‐life (t1/2) of <0.5 min, the D‐enantiomer (2) of >7 d (Fig. 2). For possible medicinal applications, the former type of derivative would be too unstable, and the latter one undesirably persistent. Thus, seven of the 256 possible ‘mixed’ Flua‐L/D‐octaarginine amides, 4a–4g, were synthesized and shown to have half‐lives in heparine‐stabilized human plasma between 8 min and 5.5 h (Figs. 3 and 4). The cell penetration of the new OAs was investigated with ‘healthy’ and with apoptotic HEK cells (Figs. 5–8), and their interactions with phospholipid bilayers were studied, using anionic lipid vesicles (Figs. 9 and 10). There are surprisingly large differences in the rates of cell penetration and binding to vesicle walls between the various stereoisomeric octaarginine derivatives 1, 2, and 4a–4g (Figs. 5 and 7). – The role of D‐amino acids and D‐peptides in nature and in drug design is briefly discussed and referenced.

Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation.

We introduce a stable isotope labeling approach for glycopeptides that allows a specific glycosylation site in a protein to be quantitatively evaluated using mass spectrometry. Succinic anhydride is used to specifically label primary amino groups of the peptide portion of the glycopeptides. The heavy form (D4(13)C4) provides an 8 Da mass increment over the light natural form (H4(12)C4), allowing simultaneous analysis and direct comparison of two glycopeptide profiles in a single MS scan. We have optimized a protocol for an in-solution trypsin digestion, a one-pot labeling procedure, and a post-labeling solid-phase extraction to obtain purified and labeled glycopeptides. We provide the first demonstration of this approach by comparing IgG1 Fc glycopeptides from polyclonal IgG samples with respect to their galactosylation and sialylation patterns using MALDI MS and LC-ESI-MS.