Petra S. Dittrich

A New Embedded Process for Compartmentalized Cell‐Free Protein Expression and On‐line Detection in Microfluidic Devices

During the last century, an enormous number of protein functions were identified, many of which can be generally described as catalytic. Ongoing research currently focuses on two aspects, analysis of the native proteome to reveal new cellular functions, and adaptation of proteins with known functions to technical processes, with the additional aspect of how these functions could be modified or improved. To this end, directed evolution of proteins has in many cases proved to be a successful strategy for designing new biocatalysts for chemical, pharmaceutical or even household use. It relies on the sensitive detection of mutants with new or improved properties, as well as their efficient singling out and amplification. In this work, a combined approach based on ultrasensitive spectroscopy, microfluidic chips and artificial cells is provided, in order to contribute a new analytical tool for both proteome discovery and evolutionary biotechnology. What is the artificial cell concept? Artificial cells reduce the features of a complex microbiological organism down to the two basic properties that are needed for defined protein analysis: compartmentalization and in vitro protein expression. A promising approach for compartmentalization in miniature environments is the formation of a water-in-oil emulsion, in which micrometer-sized water droplets embedded in a hydrophobic layer serve as artificial biocontainers. All essential compounds for transcription and translation, including the protein-encoding gene as well as a substrate to probe for catalysis, can be included within the same compartment, thus providing an essential requirement for in vitro evolution by linking genotype and phenotype. However, with classical methods for the generation of emulsions in batch (e.g. magnetic stir bar), individual droplets cannot be accurately addressed, and automated droplet handling, analysis and sorting is limited. For in vitro protein evolution, tools for automated highthroughput formation, analysis and isolation of individual biocontainers are highly desirable. 10] Therefore downscaling and integration of all functional steps on a single microchip represents an exciting advance. In microfluidic channel networks, the formation of water-in-oil emulsions at high rates and with monodisperse droplets becomes feasible, while small sample quantities can be controllably introduced, while automated droplet handling, sensitive detection of products and potential isolation of individual droplets can be performed. In this work, we demonstrate the efficient implementation of microstructured devices to generate water-in-oil emulsions, and at the same time perform in vitro expression of proteins inside the water droplets (Figure 1). By using the red-shifted

Chemical and biological single cell analysis

Single cells represent the minimal functional unit of life. A major goal of biology is to understand the mechanisms operating in this minimal unit. Nowadays, analysis of the single cell can be performed at unprecedented resolution using new lab-on-a-chip devices and advanced analytical methods. While cell handling and cultivation devices can be classified into finite volume reactors and flow systems, the analytical approaches differ in respect to invasive (i.e. chemical) and noninvasive (i.e. biological/living cell) analysis. Using these new and exciting technologies cell-to-cell differences, originating from regulatory circuits and distinct microenvironments, can now be explored. For example, it could be shown that the rates of transcription and translation are stochastic. Chemical and biological single cell analyses provide an unprecedented access to the understanding of cell-to-cell differences and basic biological concepts.

Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states.

Green fluorescent protein (GFP) from jellyfish Aequorea victoria, the powerful genetically encoded tag presently available in a variety of mutants featuring blue to yellow emission, has found a red-emitting counterpart. The recently cloned red fluorescent protein DsRed, isolated from Discosoma corals (), with its emission maximum at 583 nm, appears to be the long awaited tool for multi-color applications in fluorescence-based biological research. Studying the emission dynamics of DsRed by fluorescence correlation spectroscopy (FCS), it can be verified that this protein exhibits strong light-dependent flickering similar to what is observed in several yellow-shifted mutants of GFP. FCS data recorded at different intensities and excitation wavelengths suggest that DsRed appears under equilibrated conditions in at minimum three interconvertible states, apparently fluorescent with different excitation and emission properties. Light absorption induces transitions and/or cycling between these states on time scales of several tens to several hundreds of microseconds, dependent on excitation intensity. With increasing intensity, the emission maximum of the static fluorescence continuously shifts to the red, implying that at least one state emitting at longer wavelength is preferably populated at higher light levels. In close resemblance to GFP, this light-induced dynamic behavior implies that the chromophore is subject to conformational rearrangements upon population of the excited state.

Gene delivery with bisphosphonate-stabilized calcium phosphate nanoparticles

Nucleic acid drugs are promising new therapeutics, due to their possible applications in a wide variety of diseases and their strong targeting potential and associated lower off-target effects compared to conventional pharmaceuticals. However, their poor intracellular bioavailability and rapid degradation hinder their development as drugs. Therefore, efficient delivery is a major challenge. Various systems have been developed to overcome this problem. The entrapment of genetic material into nanoparticles constitutes a promising approach to increase the in vitro and in vivo transfection activity. Calcium phosphate-DNA co-precipitates have been used for gene delivery for more than 35 years and have the advantage of being nontoxic, easy to produce, and having the ability to complex nucleic acids leading to efficient transfection. Conventional synthetic methods yield particles that are only stable for a short period of time. Herein is proposed a versatile, surfactant-free method to stabilize calcium phosphate-DNA nanoparticles based on the use of poly(ethylene glycol)-functionalized bisphosphonate. The strength of the interaction between the bisphosphonate and the calcium phosphate enabled the formation of stable, but bioresorbable particles of around 200 nm, which exhibited physical stability over several days. Additionally, the nanoparticles revealed good and sustained ability to transfect cells while displaying low toxicity.

Breakdown of Axonal Synaptic Vesicle Precursor Transport by Microglial Nitric Oxide

The mechanism of axonal injury in inflammatory brain diseases is still unclear. Increased microglial production of nitric oxide (NO) is a common early sign in neuroinflammatory diseases. We found by fluorescence correlation spectroscopy that synaptophysin tagged with enhanced green fluorescence protein (synaptophysin-EGFP) moves anterogradely in axons of cultured neurons. Activated microglia focally inhibited the axonal movement of synaptophysin-EGFP in a NO synthase-dependent manner. Direct application of a NO donor to neurons resulted in inhibition of axonal transport of synaptophysin-EGFP and synaptotagmin I tagged with EGFP, mediated via phosphorylation of c-jun NH(2)-terminal kinase (JNK). Thus, overt production of reactive NO by activated microglia blocks the axonal transport of synaptic vesicle precursors via phosphorylation of JNK and could cause axonal and synaptic dysfunction.

Coordination Polymer Nanofibers Generated by Microfluidic Synthesis

One-dimensional coordination polymer nanostructures are an emerging class of nanoscale materials with many potential applications. Here, we report the first case of coordination polymer nanofibers assembled using microfluidic technologies. Unlike common synthetic procedures, this approach enables parallel synthesis with an unprecedented level of control over the coordination pathway and facilitates the formation of 1D coordination polymer assemblies at the nanometer length scale. Finally, these nanostructures, which are not easily constructed with traditional methods, can be used for various applications, for example as templates to grow and organize functional inorganic nanoparticles.

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.

Accessing Molecular Dynamics in Cells by Fluorescence Correlation Spectroscopy.

Abstract Fluorescence correlation spectroscopy (FCS) analyzes spontaneous fluctuations in the fluorescence emission of small molecular ensembles, thus providing information about a multitude of parameters, such as concentrations, molecular mobility and dynamics of fluorescently labeled molecules. Performed within diffractionlimited confocal volume elements, FCS provides an attractive alternative to photobleaching recovery methods for determining intracellular mobility parameters of very low quantities of fluorophores. Due to its high sensitivity sufficient for single molecule detection, the method is subject to certain artifact hazards that must be carefully controlled, such as photobleaching and intramolecular dynamics, which introduce fluorescence flickering. Furthermore, if molecular mobility is to be probed, nonspecific interactions of the labeling dye with cellular structures can introduce systematic errors. In cytosolic measurements, lipophilic dyes, such as certain rhodamines that bind to intracellular membranes, should be avoided. To study free diffusion, genetically encoded fluorescent labels such as green fluorescent protein (GFP) or DsRed are preferable since they are less likely to nonspecifically interact with cellular substructures.

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.

Droplet microfluidics with magnetic beads: a new tool to investigate drug-protein interactions

In this study, we give the proof of concept for a method to determine binding constants of compounds in solution. By implementing a technique based on magnetic beads with a microfluidic device for segmented flow generation, we demonstrate, for individual droplets, fast, robust and complete separation of the magnetic beads. The beads are used as a carrier for one binding partner and hence, any bound molecule is separated likewise, while the segmentation into small microdroplets ensures fast mixing, and opens future prospects for droplet-wise analysis of drug candidate libraries. We employ the method for characterization of drug–protein binding, here warfarin to human serum albumin. The approach lays the basis for a microfluidic droplet-based screening device aimed at investigating the interactions of drugs with specific targets including enzymes and cells. Furthermore, the continuous method could be employed for various applications, such as binding assays, kinetic studies, and single cell analysis, in which rapid removal of a reactive component is required.