Simon Kuster

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.

High-resolution droplet-based fractionation of nano-LC separations onto microarrays for MALDI-MS analysis.

We present a robust droplet-based device, which enables the fractionation of ultralow flow rate nanoflow liquid chromatography (nano-LC) eluate streams at high frequencies and high peak resolution. This is achieved by directly interfacing the separation column to a micro T-junction, where the eluate stream is compartmentalized into picoliter droplets. This immediate compartmentalization prevents peak dispersion during eluate transport and conserves the chromatographic performance. Subsequently, nanoliter eluate fractions are collected at a rate of one fraction per second on a high-density microarray to retain the separation with high temporal resolution. Chromatographic separations of up to 45 min runtime can thus be archived on a single microarray possessing 2700 sample spots. The performance of this device is demonstrated by fractionating the separation of a tryptic digest of a known protein mixture onto the microarray chip and subsequently analyzing the sample archive using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Resulting peak widths are found to be significantly reduced compared to standard continuous flow spotting technologies as well as in comparison to a conventional nano-LC-electrospray ionization-mass spectrometry interface. Moreover, we demonstrate the advantage of our high-definition nanofractionation device by applying two different MALDI matrices to all collected fractions in an alternating fashion. Since the information that is obtained from a MALDI-MS measurement depends on the choice of MALDI matrix, we can extract complementary information from neighboring spots containing almost identical composition but different matrices.

Self-Aliquoting Microarray Plates for Accurate Quantitative Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a fast analysis tool employed for the detection of a broad range of analytes. However, MALDI-MS has a reputation of not being suitable for quantitative analysis. Inhomogeneous analyte/matrix co-crystallization, spot-to-spot inhomogeneity, as well as a typically low number of replicates are the main contributing factors. Here, we present a novel MALDI sample target for quantitative MALDI-MS applications, which addresses the limitations mentioned above. The platform is based on the recently developed microarray for mass spectrometry (MAMS) technology and contains parallel lanes of hydrophilic reservoirs. Samples are not pipetted manually but deposited by dragging one or several sample droplets with a metal sliding device along these lanes. Sample is rapidly and automatically aliquoted into the sample spots due to the interplay of hydrophilic/hydrophobic interactions. With a few microliters of sample, it is possible to aliquot up to 40 replicates within seconds, each aliquot containing just 10 nL. The analyte droplet dries immediately and homogeneously, and consumption of the whole spot during MALDI-MS analysis is typically accomplished within few seconds. We evaluated these sample targets with respect to their suitability for use with different samples and matrices. Furthermore, we tested their application for generating calibration curves of standard peptides with α-cyano-4-hdydroxycinnamic acid as a matrix. For angiotensin II and [Glu(1)]-fibrinopeptide B we achieved coefficients of determination (r(2)) greater than 0.99 without the use of internal standards.

Synthesis and characterization of organic dyes with various electron-accepting substituents for p-type dye-sensitized solar cells

Four new donor-π-acceptor dyes differing in their acceptor group have been synthesized and employed as model systems to study the influence of the acceptor groups on the photophysical properties and in NiO-based p-type dye-sensitized solar cells. UV/Vis absorption spectra showed a broad range of absorption coverage with maxima between 331 and 653 nm. Redox potentials as well as HOMO and LUMO energies of the dyes were determined from cyclic voltammetry measurements and evaluated concerning their potential use as sensitizers in p-type dye-sensitized solar cells (p-DSCs). Quantum-chemical density functional theory calculations gave further insight into the frontier orbital distributions, which are relevant for the electronic processes in p-DSCs. In p-DSCs using an iodide/triiodide-based electrolyte, the polycyclic 9,10-dicyano-acenaphtho[1,2-b]quinoxaline (DCANQ) acceptor-containing dye gave the highest power conversion efficiency of 0.08%, which is comparable to that obtained with the perylenemonoimide (PMI)-containing dye. Interestingly, devices containing the DCANQ-based dye achieve a higher V(OC) of 163 mV compared to 158 mV for the PMI-containing dye. The result was further confirmed by impedance spectroscopic analysis showing higher recombination resistance and thus a lower recombination rate for devices containing the DCANQ dye than for PMI dye-based devices. However, the use of the strong electron-accepting tricyanofurane (TCF) group played a negative role in the device performance, yielding an efficiency of only 0.01% due to a low-lying LUMO energy level, thus resulting in an insufficient driving force for efficient dye regeneration. The results demonstrate that a careful molecular design with a proper choice of the acceptor unit is essential for development of sensitizers for p-DSCs.

A Microarray-Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Approach for Site-specific Protein N-glycosylation Analysis, as Demonstrated for Human Serum Immunoglobulin M (IgM)

We demonstrate a new approach for the site-specific identification and characterization of protein N-glycosylation. It is based on a nano-liquid chromatography microarray-matrix assisted laser desorption/ionization-MS platform, which employs droplet microfluidics for on-plate nanoliter reactions. A chromatographic separation of a proteolytic digest is deposited at a high frequency on the microarray. In this way, a short separation run is archived into thousands of nanoliter reaction cavities, and chromatographic peaks are spread over multiple array spots. After fractionation, each other spot is treated with PNGaseF to generate two correlated traces within one run, one with treated spots where glycans are enzymatically released from the peptides, and one containing the intact glycopeptides. Mining for distinct glycosites is performed by searching for the predicted deglycosylated peptides in the treated trace. An identified peptide then leads directly to the position of the “intact” glycopeptide clusters, which are located in the adjacent spots. Furthermore, the deglycosylated peptide can be sequenced efficiently in a simple collision-induced dissociation-MS experiment. We applied the microarray approach to a detailed site-specific glycosylation analysis of human serum IgM. By scanning the treated spots with low-resolution matrix assisted laser desorption/ionization-time-of-flight-MS, we observed all five deglycosylated peptides, including the one originating from the secretory chain. A detailed glycopeptide characterization was then accomplished on the adjacent, untreated spots with high mass resolution and high mass accuracy using a matrix assisted laser desorption ionization-Fourier transform-MS. We present the first detailed and comprehensive mass spectrometric analysis on the glycopeptide level for human polyclonal IgM with high mass accuracy. Besides complex type glycans on Asn 395, 332, 171, and on the J chain, we observed oligomannosidic glycans on Asn 563, Asn 402 and minor amounts of oligomannosidic glycans on the glycosite Asn 171. Furthermore, hybrid type glycans were found on Asn 402, Asn 171 and in traces Asn 332.

Transient receptor potential vanilloid 2-mediated shear-stress responses in C2C12 myoblasts are regulated by serum and extracellular matrix

The developmental sensitivity of skeletal muscle to mechanical forces is unparalleled in other tissues. Calcium entry via reputedly mechanosensitive transient receptor potential (TRP) channel classes has been shown to play an essential role in both the early proliferative stage and subsequent differentiation of skeletal muscle myoblasts, particularly TRP canonical (TRPC) 1 and TRP vanilloid (TRPV) 2. Here we show that C2C12 murine myoblasts respond to fluid flow‐induced shear stress with increments in cytosolic calcium that are largely initiated by the mechanosensitive opening of TRPV2 channels. Response to fluid flow was augmented by growth in low extracellular serum concentration (5 vs. 20% fetal bovine serum) by greater than 9‐fold and at 18 h in culture, coincident with the greatest TRPV2 channel expression under identical conditions (P < 0.02). Fluid flow responses were also enhanced by substrate functionalization with laminin, rather than with fibronectin, agreeing with previous findings that the gating of TRPV2 is facilitated by laminin. Fluid flow‐induced calcium increments were blocked by ruthenium red (27%) and SKF‐96365 (38%), whereas they were unaltered by 2‐aminoethoxydiphenyl borate, further corroborating that TRPV2 channels play a predominant role in fluid flow mechanosensitivity over that of TRPC1 and TRP melastatin (TRPM) 7.—Kurth, F., Franco‐Obregón, A., Casarosa, M., Küster, S. K., Wuertz‐Kozak, K., Dittrich, P. S. Transient receptor potential vanilloid 2‐mediated shear‐stress responses in C2C12 myoblasts are regulated by serum and extracellular matrix. FASEB J. 29, 4726‐4737 (2015). www.fasebj.org

Screening for protein phosphorylation using nanoscale reactions on microdroplet arrays

We present a novel and straightforward screening method to detect protein phosphorylations in complex protein mixtures. A proteolytic digest is separated by a conventional nanoscale liquid chromatography (nano-LC) separation and the eluate is immediately compartmentalized into microdroplets, which are spotted on a microarray MALDI plate. Subsequently, the enzyme alkaline phosphatase is applied to every second microarray spot to remove the phosphate groups from phosphorylated peptides, which results in a mass shift of n×-80 Da. The MALDI-MS scan of the microarray is then evaluated by a software algorithm to automatically identify the phosphorylated peptides by exploiting the characteristic chromatographic peak profile induced by the phosphatase treatment. This screening method does not require extensive MS/MS experiments or peak list evaluation and can be easily extended to other enzymatic or chemical reactions.

Magnetically Enhanced Bicelles Delivering Switchable Anisotropy in Optical Gels

Mesostructures responding to external triggers such as temperature, pH, or magnetic field have the potential to be used as self-acting sensors, detectors, or switches. Key features are a strong and well-defined response to the external trigger. Here, we present magnetic alignable bicelles embedded into a gelatin matrix generating magnetically switchable structures, which can reversibly be locked and unlocked by adjusting the temperature. We show that the disk-like aggregates can be orientated in magnetic fields, and such orientation can be preserved after embedding into gelatin. The resulting gel cubes show an anisotropic transfer for electromagnetic waves, i.e., a different spatial birefringence. Cycling through the melting point of gelatin sets the structure back to its isotropic state providing a read-out of the thermal history. Stacking of the bicelles induced by the gelatin promotes magnetic aligning, as an increased aggregation number in the stacks increases the magnetic orientation energy.

Hagfish slime and mucin flow properties and their implications for defense

When hagfish (Myxinidae) are attacked by predators, they form a dilute, elastic, and cohesive defensive slime made of mucins and protein threads. In this study we propose a link between flow behavior and defense mechanism of hagfish slime. Oscillatory rheological measurements reveal that hagfish slime forms viscoelastic networks at low concentrations. Mucins alone did not contribute viscoelasticity, however in shear flow, viscosity was observed. The unidirectional flow, experienced by hagfish slime during suction feeding by predators, was mimicked with extensional rheology. Elongational stresses were found to increase mucin viscosity. The resulting higher resistance to flow could support clogging of the attacker’s gills. Shear flow in contrast decreases the slime viscosity by mucin aggregation and leads to a collapse of the slime network. Hagfish may benefit from this collapse when trapped in their own slime and facing suffocation by tying a sliding knot with their body to shear off the slime. This removal could be facilitated by the apparent shear thinning behavior of the slime. Therefore hagfish slime, thickening in elongation and thinning in shear, presents a sophisticated natural high water content gel with flow properties that may be beneficial for both, defense and escape.