Development of a fully integrated falling film microreactor for gas-liquid-solid biotransformation with surface immobilized O2 -dependent enzyme.

Microstructured flow reactors are powerful tools for the development of multiphase biocatalytic transformations. To expand their current application also to O2 -dependent enzymatic conversions, we have implemented a fully integrated falling film microreactor that provides controllable countercurrent gas-liquid phase contacting in a multi-channel microstructured reaction plate. Advanced non-invasive optical sensing is applied to measure liquid-phase oxygen concentrations in both in- and out-flow as well as directly in the microchannels (width: 600 µm; depth: 200 µm). Protein-surface interactions are designed for direct immobilization of catalyst on microchannel walls. Target enzyme (here: d-amino acid oxidase) is fused to the positively charged mini-protein Zbasic2 and the channel surface contains a negatively charged γ-Al2 O3 wash-coat layer. Non-covalent wall attachment of the chimeric Zbasic2 _oxidase resulted in fully reversible enzyme immobilization with fairly uniform surface coverage and near complete retention of biological activity. The falling film at different gas and liquid flow rates as well as reactor inclination angles was shown to be mostly wavy laminar. The calculated film thickness was in the range 0.5-1.3 × 10(-4) m. Direct O2 concentration measurements at the channel surface demonstrated that the liquid side mass transfer coefficient (KL ) for O2 governed the overall gas/liquid/solid mass transfer and that the O2 transfer rate (≥0.75 mM · s(-1) ) vastly exceeded the maximum enzymatic reaction rate in a wide range of conditions. A value of 7.5 (±0.5) s(-1) was determined for the overall mass transfer coefficient KL a, comprising a KL of about 7 × 10(-5) m · s(-1) and a specific surface area of up to 10(5) m(-1) . Biotechnol. Bioeng. 2016;113: 1862-1872. © 2016 Wiley Periodicals, Inc.

A microfluidically perfused three dimensional human liver model.

Within the liver, non-parenchymal cells (NPCs) are critically involved in the regulation of hepatocyte polarization and maintenance of metabolic function. We here report the establishment of a liver organoid that integrates NPCs in a vascular layer composed of endothelial cells and tissue macrophages and a hepatic layer comprising stellate cells co-cultured with hepatocytes. The three-dimensional liver organoid is embedded in a microfluidically perfused biochip that enables sufficient nutrition supply and resembles morphological aspects of the human liver sinusoid. It utilizes a suspended membrane as a cell substrate mimicking the space of Disse. Luminescence-based sensor spots were integrated into the chip to allow online measurement of cellular oxygen consumption. Application of microfluidic flow induces defined expression of ZO-1, transferrin, ASGPR-1 along with an increased expression of MRP-2 transporter protein within the liver organoids. Moreover, perfusion was accompanied by an increased hepatobiliary secretion of 5(6)-carboxy-2',7'-dichlorofluorescein and an enhanced formation of hepatocyte microvilli. From this we conclude that the perfused liver organoid shares relevant morphological and functional characteristics with the human liver and represents a new in vitro research tool to study human hepatocellular physiology at the cellular level under conditions close to the physiological situation.

Imaging of oxygen in microreactors and microfluidic systems.

This review gives an overview on the state-of-the-art of oxygen imaging in microfluidics. Oxygen imaging using optical oxygen sensors based on luminescence is a versatile and powerful tool for obtaining profoundly space-resolved information of oxygen in microreactors and microfluidic systems. We briefly introduce the principle of oxygen imaging and present techniques of oxygen imaging applied in microreactors and microfluidic devices, including selection criteria and demands of sensing material and basic set-up for a 2D oxygen sensing system. A detailed review of oxygen imaging in microreactors and microfluidic systems is given on different applications in oxygen gradient monitoring, cell culturing, single-cell analysis and chemical reactions. Finally, we discuss challenges and trends of oxygen imaging in microfluidic systems.

Magnetic optical sensor particles: a flexible analytical tool for microfluidic devices.

In this study we evaluate magnetic optical sensor particles (MOSePs) with incorporated sensing functionalities regarding their applicability in microfluidic devices. MOSePs can be separated from the surrounding solution to form in situ sensor spots within microfluidic channels, while read-out is accomplished outside the chip. These magnetic sensor spots exhibit benefits of sensor layers (high brightness and convenient usage) combined with the advantages of dispersed sensor particles (ease of integration). The accumulation characteristics of MOSePs with different diameters were investigated as well as the in situ sensor spot stability at varying flow rates. Magnetic sensor spots were stable at flow rates specific to microfluidic applications. Furthermore, MOSePs were optimized regarding fiber optic and imaging read-out systems, and different referencing schemes were critically discussed on the example of oxygen sensors. While the fiber optic sensing system delivered precise and accurate results for measurement in microfluidic channels, limitations due to analyte consumption were found for microscopic oxygen imaging. A compensation strategy is provided, which utilizes simple pre-conditioning by exposure to light. Finally, new application possibilities were addressed, being enabled by the use of MOSePs. They can be used for microscopic oxygen imaging in any chip with optically transparent covers, can serve as flexible sensor spots to monitor enzymatic activity or can be applied to form fixed sensor spots inside microfluidic structures, which would be inaccessible to integration of sensor layers.

Fluorescent materials for pH sensing and imaging based on novel 1,4-diketopyrrolo-[3,4-c]pyrrole dyes†Electronic supplementary information (ESI) available: NMR and MS spectra, further sensor characteristics and sensor long-time performance. See DOI: 10.1039/c3tc31130aClick here for additional data file.

New optical pH-sensors relying on 1,4-diketopyrrolo-[3,4-c]pyrroles (DPPs) as fluorescent pH-indicators are presented. Different polymer hydrogels are useful as immobilization matrices, achieving excellent sensitivity and good brightness in the resulting sensor. The operational pH can be tuned over a wide range (pH 5-12) by selecting the fine structure of the indicator and the matrix. A ratiometric sensor in the form of nanoparticles is also presented. It is suitable for RGB camera readout, and its practical applicability for fluorescence imaging in microfluidic systems is demonstrated. The indicators are synthesized starting from the commercially available DPP pigments by a straightforward concept employing chlorosulfonation and subsequent reaction with amines. Their sensitivity derives from two distinct mechanisms. At high pH (>9), they exhibit a remarkable alteration of both absorption and fluorescence spectra due to deprotonation of the lactam nitrogen atoms. If a phenolic group is introduced, highly effective fluorescence quenching at near-neutral pH occurs due to photoinduced electron transfer (PET) involving the phenolate form.

Microfluidic oxygen imaging using integrated optical sensor layers and a color camera.

In this work we present a high resolution oxygen imaging approach, which can be used to study 2D oxygen distribution inside microfluidic environments. The presented setup comprises a fabrication process of microfluidic chips with integrated luminescent sensing films combined with referenced oxygen imaging applying a color CCD-camera. Enhancement of the sensor performance was achieved by applying the principle of light harvesting. This principle enabled ratiometric imaging employing the red and the green channel of a color CCD-camera. The oxygen sensitive emission of platinum(ii)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorphenyl)-porphyrin (PtTFPP) was detected by the red channel, while the emission of a reference dye was detected by the green channel. This measurement setup allowed for accurate real-time 2D oxygen imaging with superior quality compared to intensity imaging. The sensor films were subsequently used to measure the respiratory activity of human cell cultures (HeLa carcinoma cells and normal human dermal fibroblasts) in a microfluidic system. The sensor setup is well suited for different applications from spatially and temporally resolving oxygen concentration inside microfluidic channels to parallelization of oxygen measurements and paves the way to novel cell based assays, e.g. in tissue engineering, tumor biology and hypoxia reperfusion phenomena.

Fast responsive, optical trace level ammonia sensor for environmental monitoring.

BACKGROUND: Ammonia is a ubiquitous chemical substance which is created in technical and biological processes and harmful to many different organisms. One specific problem is the toxicity of ammonia in fish at levels of 25 µg/l - a very common issue in today's aqua culture. In this study we report a development of a fast responsive, optical ammonia sensor for trace concentrations. RESULTS: Different hydrogels have been investigated as host polymers for a pH based sensing mechanism based on fluorescent dyes. A porous hydrophobic fluoropolymer membrane was used as an ion barrier cover layer to achieve a good ammonia permeability. The sensor's sensitivity towards ammonia as well as crosssensitivity towards pH-value and salinity, and the temperature dependency have been determined. Two different methods to reference fluorescence signals have been employed to eliminate intensity-based measurement drawbacks. CONCLUSION: The presented sensor features high sensitivity and a fast response even at concentrations near 1 ppb. No cross sensitivity towards pH and salinity could be observed and temperature dependency was determined as compensateable. Both referencing approaches prove themselves to be able to provide a simple use of the sensor for in-field applications.

Filter-free integrated sensor array based on luminescence and absorbance measurements using ring-shaped organic photodiodes.

An optical waveguiding sensor array featuring monolithically integrated organic photodiodes as integrated photo-detector, which simplifies the readout system by minimizing the required parts, is presented. The necessity of any optical filters becomes redundant due to the proposed platform geometry, which discriminates between excitation light and sensing signal. The sensor array is capable of measuring luminescence or absorption, and both sensing geometries are based on the identical substrate. It is demonstrated that background light is virtually non-existent. All sensing and waveguide layers, as well as in- and out-coupling elements are assembled by conventional screen-printing techniques. Organic photodiodes are integrated by layer-by-layer vacuum deposition onto glass or common polymer foils. The universal and simple applicability of this sensor chip is demonstrated by sensing schemes for four different analytes. Relative humidity, oxygen, and carbon dioxide are measured in gas phase using luminescence-based sensor schemes; the latter two analytes are also measured by absorbance-based sensor schemes. Furthermore, oxygen and pH in aqueous media were enabled. The consistency of calibration characteristics extending over different sensor chips is verified.