Downscaling screening cultures in a multifunctional bioreactor array-on-a-chip for speeding up optimization of yeast-based lactic acid bioproduction.

A key challenge for bioprocess engineering is the identification of the optimum process conditions for the production of biochemical and biopharmaceutical compounds using prokaryotic as well as eukaryotic cell factories. Shake flasks and bench-scale bioreactor systems are still the golden standard in the early stage of bioprocess development, though they are known to be expensive, time-consuming, and labor-intensive as well as lacking the throughput for efficient production optimizations. To bridge the technological gap between bioprocess optimization and upscaling, we have developed a microfluidic bioreactor array to reduce time and costs, and to increase throughput compared with traditional lab-scale culture strategies. We present a multifunctional microfluidic device containing 12 individual bioreactors (Vt = 15 µl) in a 26 mm × 76 mm area with in-line biosensing of dissolved oxygen and biomass concentration. Following initial device characterization, the bioreactor lab-on-a-chip was used in a proof-of-principle study to identify the most productive cell line for lactic acid production out of two engineered yeast strains, evaluating whether it could reduce the time needed for collecting meaningful data compared with shake flasks cultures. Results of the study showed significant difference in the strains' productivity within 3 hr of operation exhibiting a 4- to 6-fold higher lactic acid production, thus pointing at the potential of microfluidic technology as effective screening tool for fast and parallelizable industrial bioprocess development.

Optical ammonia sensors based on fluorescent aza-BODIPY dyes- a flexible toolbox.

We present three types of optical ammonia sensors suitable for environmental, bioprocess, and reaction monitoring. A respective fluorescent BF2-chelated tetraarylazadipyrromethene dye (aza-BODIPYs) is physically entrapped in a polyurethane hydrogel (HydroMed D4) forming an emulsion system with vinyl-terminated polydimethylsiloxane (PDMS). The analyte-sensitive layer is covered by a hydrophobic membrane which excludes hydrophilic substances. Three different protection layers are tested, whereby the Teflon and the hydrophobic PES layers outperform a PDMS/TiO2 layer. Response times within their dynamic range of 15 s can be achieved, whereas the PDMS/TiO2-covered sensor requires at least 390 s. The three sensors entail the following concentration areas: first sensor 3 µg L-1-3 mg L-1 (LOD 0.23 µg L-1), second sensor 0.1-30 mg L-1 (LOD 28 µg L-1), and third sensor 3 mg L-1-1 g L-1 (LOD 0.51 mg L-1). Readout is performed with a commercially available phase fluorimeter combined with optical fibers. Dual-lifetime referencing (DLR) is used as referencing method and Egyptian blue acts as an inert reference material. No cross-sensitivity to pH changes can be detected.

A fully online sensor-equipped, disposable multiphase microbioreactor as a screening platform for biotechnological applications.

A new disposable, multiphase, microbioreactor (MBR; with a working volume of 550 µl) equipped with online sensors is presented for biotechnological screening research purposes owing to its high-throughput potential. Its design and fabrication, online sensor integration, and operation are described. During aerobic cultivation, sufficient oxygen supply is the most important factor that influences growth and product formation. The MBR is a microbubble column bioreactor (µBC), and the oxygen supply was realized by active pneumatic bubble aeration, ensuring sufficient volumetric liquid-phase mass transfer (k L a) and proper homogenization of the cultivation broth. The µBC was equipped with miniaturized sensors for the pH, dissolved oxygen, optical density and glucose concentration that allowed real-time online monitoring of these process variables during cultivation. The challenge addressed here was the integration of sensors in the limited available space. The MBR was shown to be a suitable screening platform for the cultivation of biological systems. Batch cultivations of Saccharomyces cerevisiae were performed to observe the variation in the process variables over time and to show the robustness and operability of all the online sensors in the MBR.

Trace Ammonia Sensors Based on Fluorescent Near-Infrared-Emitting aza-BODIPY Dyes.

Highly sensitive ammonia sensors for environmental monitoring are presented. The sensing materials are based on fluorescent BF2-chelated tetraarylazadipyrromethene dyes (aza-BODIPYs) dyes physically entrapped in polyurethane hydrogels and dispersed in silicone rubber. This layer is covered by a hydrophobic porous Teflon membrane used as an additional proton barrier and light scattering layer. The dual-lifetime referenced (DLR) sensors make use of near-infrared (NIR)-emitting Egyptian blue as a reference material and in combination with optical fibers are read-out via a compact phase-fluorometer. The detectable concentration range can be tuned by the choice of aza-BODIPY dye or/and the hydrogel matrix. The most sensitive sensor has a limit of detection (LOD) of 0.11 µg/L and the upper detectable concentration of 300 µg/L. No cross-sensitivity toward pH is observed. The sensors show remarkable operational stability with no noticeable drift over a period of 2 weeks.

Lifetime of Phosphorescence from Nanoparticles Yields Accurate Measurement of Concentration of Oxygen in Microdroplets, Allowing One To Monitor the Metabolism of Bacteria.

A method to monitor the level of oxygen in microdroplets is presented. Optical sensor nanoparticles are dispersed in the aqueous phase of the microfluidic droplets for culturing bacteria. The oxygen sensor nanoparticles consist of phosphorescent indicator dye embedded in poly(styrene-block-vinylpyrrolidone) nanobeads. The nanoparticles are excitable by red light and emit in the near-infrared spectra region which minimizes background fluorescence from biological matter. The biocompatibility of the nanoparticles was proven. Nanoparticles sensors were read out by adapted miniaturized oxygen meters. The instruments can be easily integrated into the microfluidic system by placing it next to the tubing and measuring through the tubing wall. The phosphorescence lifetime-based measurement circumvents the drawbacks of intensity-based measurements and enables the determination of the absolute oxygen concentration in individual moving droplets. The technique can also be used for monitoring the growth of bacteria in microdroplets. We demonstrate simultaneous measurement of concentration of oxygen and optical density (OD) from micro cultures of E. coli and M. smegmatis.

Simultaneous Determination of Oxygen and pH Inside Microfluidic Devices Using Core-Shell Nanosensors.

A powerful online analysis setup for the simultaneous detection of oxygen and pH is presented. It features core-shell nanosensors, which enable contactless and inexpensive read-out using adapted oxygen meters via modified dual lifetime referencing in the frequency domain (phase shift measurements). Lipophilic indicator dyes were incorporated into core-shell structured poly(styrene-block-vinylpyrrolidone) nanoparticles (average diameter = 180 nm) yielding oxygen nanosensors and pH nanosensors by applying different preparation protocols. The oxygen indicator platinum(II) meso-tetra(4-fluorophenyl) tetrabenzoporphyrin (PtTPTBPF) was entrapped into the polystyrene core (oxygen nanosensors) and a pH sensitive BF2-chelated tetraarylazadipyrromethene dye (aza-BODIPY) was incorporated into the polyvinylpyrrolidone shell (pH nanosensors). The brightness of the pH nanoparticles was increased by more than 3 times using a light harvesting system. The nanosensors have several advantages such as being excitable with red light, emitting in the near-infrared spectral region, showing a high stability in aqueous media even at high particle concentrations, high ionic strength, or high protein concentrations and are spectrally compatible with the used read-out device. The resolution for oxygen of the setup is 0.5-2.0 hPa (approximately 0.02-0.08 mg/L of dissolved oxygen) at low oxygen concentrations (<50 hPa) and 4-8 hPa (approximately 0.16-0.32 mg/L of dissolved oxygen) at ambient air oxygen concentrations (approximately 200 hPa at 980 mbar air pressure) at room temperature. The pH resolution is 0.03-0.1 pH units within the dynamic range (apparent pKa 7.23 ± 1.0) of the nanosensors. The sensors were used for online monitoring of pH changes during the enzymatic transformation of Penicillin G to 6-aminopenicillanic acid catalyzed by Penicillin G acylase in miniaturized stirred batch reactors or continuous flow microreactors.

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.

Measurement of oxygen transfer from air into organic solvents.

BACKGROUND: The use of non-aqueous organic media is becoming increasingly important in many biotechnological applications in order to achieve process intensification. Such media can be used, for example, to directly extract poorly water-soluble toxic products from fermentations. Likewise many biological reactions require the supply of oxygen, most normally from air. However, reliable online measurements of oxygen concentration in organic solvents (and hence oxygen transfer rates from air to the solvent) has to date proven impossible due to limitations in the current analytical methods. RESULTS: For the first time, online oxygen measurements in non-aqueous media using a novel optical sensor are demonstrated. The sensor was used to measure oxygen concentration in various organic solvents including toluene, THF, isooctane, DMF, heptane and hexane (which have all been shown suitable for several biological applications). Subsequently, the oxygen transfer rates from air into these organic solvents were measured. CONCLUSION: The measurement of oxygen transfer rates from air into organic solvents using the dynamic method was established using the solvent resistant optical sensor. The feasibility of online oxygen measurements in organic solvents has also been demonstrated, paving the way for new opportunities in process control. © 2015 The Authors. Journal of Chemical Technology & Biotechnology published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry.

MicroRNA-26 family is required for human adipogenesis and drives characteristics of brown adipocytes.

Adipose tissue contains thermogenic adipocytes (i.e., brown and brite/beige) that oxidize nutrients at exceptionally high rates via nonshivering thermogenesis. Its recent discovery in adult humans has opened up new avenues to fight obesity and related disorders such as diabetes. Here, we identified miR-26a and -26b as key regulators of human white and brite adipocyte differentiation. Both microRNAs are upregulated in early adipogenesis, and their inhibition prevented lipid accumulation while their overexpression accelerated it. Intriguingly, miR-26a significantly induced pathways related to energy dissipation, shifted mitochondrial morphology toward that seen in brown adipocytes, and promoted uncoupled respiration by markedly increasing the hallmark protein of brown fat, uncoupling protein 1. By combining in silico target prediction, transcriptomics, and an RNA interference screen, we identified the sheddase ADAM metallopeptidase domain 17 (ADAM17) as a direct target of miR-26 that mediated the observed effects on white and brite adipogenesis. These results point to a novel, critical role for the miR-26 family and its downstream effector ADAM17 in human adipocyte differentiation by promoting characteristics of energy-dissipating thermogenic adipocytes.

NIR-emitting aza-BODIPY dyes--new building blocks for broad-range optical pH sensors.

New aza-BODIPY indicators which cover the pH scale from 1.5 to 13 are presented. The new indicators feature absorption/emission bands in the red/near-infrared (NIR) spectral region, exhibit high molar absorption coefficients (∼ 80,000 M(-1) cm(-1)) and show good quantum yields (∼20%). All dyes represent promising building blocks for the development of a broad-range sensor for various pH ranges. Combination of four of these pH indicators yields a pH sensor with an extended dynamic range from pH 2 to 9.

Label-free microfluidic free-flow isoelectric focusing, pH gradient sensing and near real-time isoelectric point determination of biomolecules and blood plasma fractions.

We demonstrate the fabrication, characterization and application of microfluidic chips capable of continuous electrophoretic separation via free flow isoelectric focussing (FFIEF). By integration of a near-infrared (NIR) fluorescent pH sensor layer under the whole separation bed, on-line observation of the pH gradient and determination of biomolecular isoelectric points (pI) was achieved within a few seconds. Using an optical setup for imaging of the intrinsic fluorescence of biomolecules at 266 nm excitation, labelling steps could be avoided and the native biomolecules could be separated, collected and analysed for their pI. The fabricated microchip was successfully used for the monitoring of the separation and simultaneous observation of the pH gradient during the isoelectric focussing of the proteins α-lactalbumin and ß-lactoglobulin, blood plasma proteins and the antibiotics ampicillin and ofloxacin. The obtained pIs are in good agreement with literature data, demonstrating the applicability of the system. Mass spectra from the separated antibiotics taken after 15 minutes of continuous separation from different fractions at the end of the microchip validated the separation via microfluidic isoelectric focussing and indicate the possibility of further on- or off-chip processing steps.

Low cost referenced luminescent imaging of oxygen and pH with a 2-CCD colour near infrared camera.

A low cost imaging set-up for optical chemical sensors based on NIR-emitting dyes is presented. It is based on a commercially available 2-CCD colour near infrared camera, LEDs and tailor-made optical sensing materials for oxygen and pH. The set-up extends common ratiometric RGB imaging based on the red, green and blue channels of colour cameras by an additional NIR channel. The hardware and software of the camera were adapted to perform ratiometric imaging. A series of new planar sensing foils were introduced to image oxygen, pH and both parameters simultaneously. The used NIR-emitting indicators are based on benzoporphyrins and aza-BODIPYs for oxygen and pH, respectively. Moreover, a wide dynamic range oxygen sensor is presented. It allows accurate imaging of oxygen from trace levels up to ambient air concentrations. The imaging set-up in combination with the normal range ratiometric oxygen sensor showed a resolution of 4-5 hPa at low oxygen concentrations (<50 hPa) and 10-15 hPa at ambient air oxygen concentrations; the trace range oxygen sensor (<20 hPa) revealed a resolution of about 0.5-1.8 hPa. The working range of the pH-sensor was in the physiological region from pH 6.0 up to pH 8.0 and showed an apparent pKa-value of 7.3 with a resolution of about 0.1 pH units. The performance of the dual parameter oxygen/pH sensor was comparable to the single analyte pH and normal range oxygen sensors.

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.

Microphysiological pancreas-on-chip platform with integrated sensors to model endocrine function and metabolism.

Pancreatic in vitro research is of major importance to advance mechanistic understanding and development of treatment options for diseases such as diabetes mellitus. We present a thermoplastic-based microphysiological system aiming to model the complex microphysiological structure and function of the endocrine pancreas with concurrent real-time read-out capabilities. The specifically tailored platform enables self-guided trapping of single islets at defined locations: ß-cells are assembled to pseudo-islets and injected into the tissue chamber using hydrostatic pressure-driven flow. The pseudo-islets can further be embedded in an ECM-like hydrogel mimicking the native microenvironment of pancreatic islets in vivo. Non-invasive real-time monitoring of the oxygen levels on-chip is realized by the integration of luminescence-based optical sensors to the platform. To monitor insulin secretion kinetics in response to glucose stimulation in a time-resolved manner, an automated cycling of different glucose conditions is implemented. The model's response to glucose stimulation can be monitored via offline analysis of insulin secretion and via specific changes in oxygen consumption due to higher metabolic activity of pseudo-islets at high glucose levels. To demonstrate applicability for drug testing, the effects of antidiabetic medications are assessed and changes in dynamic insulin secretion are observed in line with the respective mechanism of action. Finally, by integrating human pancreatic islet microtissues, we highlight the flexibility of the platform and demonstrate the preservation of long-term functionality of human endocrine pancreatic tissue.

Quantitating intraparticle O2 gradients in solid supported enzyme immobilizates: experimental determination of their role in limiting the catalytic effectiveness of immobilized glucose oxidase.

Enzymatic O2 -dependent oxidations are receiving increased attention for use in fine chemicals synthesis. Solid supported oxidation catalysts often show poor efficiency due to pronounced O2 diffusion restriction. Internal O2 supply therefore constitutes a key parameter for optimizing the enzyme immobilization. We herein describe an optical sensing method for quantitation of space-averaged intraparticle O2 concentrations in porous Sepabeads carriers. The method applies phosphorescence lifetime measurements on Sepabeads labeled with an O2 sensitive indicator dye. Using glucose oxidase immobilized at different loadings (0.005-12 mg/g) on labeled Sepabeads, we analyzed in real time during the enzymatic reaction the formation of O2 concentration differences between bulk liquid and the intraparticle environment. We show that the O2 gradient at apparent steady state increased with increasing enzyme loading, so that O2 eventually became totally depleted from inside the highly loaded carriers. We also show that the residual intraparticle O2 concentration was correlated with the catalytic effectiveness factor (η) of the enzyme immobilizate used, thus providing a direct measure of the magnitude of O2 diffusion limitation. Once corrected for diffusional effect, η was no longer dependent on enzyme loading and its constant value now described the intrinsic activity of immobilized glucose oxidase. Three common procedures of enzyme immobilization, involving adsorption, cross-linking, and covalent attachment, are shown to differ widely concerning the obtained intrinsic activity. Therefore, intraparticle O2 concentration data enable distinction between diffusional restriction and activity loss as the two principal factors limiting the effectiveness of immobilized O2 dependent enzymes, and thus they inform rational design of an optimally active oxidation biocatalyst on solid support.

Enabling luminescence decay time-based sensing using integrated organic photodiodes.

The use of organic photodiodes (OPDs) for measuring phosphorescent lifetimes of optochemical oxygen sensors is described. Phosphorescent indicators with lifetimes ranging from ∼5 to 60 µs have been studied using light-emitting diodes as the excitation source and organic photodiodes integrated into the sensor substrate for detection. A measurement system using an adjusted electronic circuitry to detect photocurrents in the nanoampere range is presented. The response behaviour of the organic photodiodes has been characterized, and it was found that a forward (positive) bias had to be applied in order to decrease the response time of the OPDs to a range suitable for phosphorescence decay time measurements. A modulation cutoff frequency of ∼100 kHz has been determined, corresponding to a response time of the organic photodiodes of 1.6 µs. Two sensor dyes have been characterized regarding their lifetimes upon exposure to 0-20% oxygen, and it was shown that results comparable to literature data and inorganic photodetectors can be achieved.

Optical glucose sensor for microfluidic cell culture systems.

Glucose is the primary energy source of human cells. Therefore, monitoring glucose inside microphysiological systems (MPS) provides valuable information on the viability and metabolic state of the cultured cells. However, continuous glucose monitoring inside MPS is challenging due to a lack of suitable miniaturized sensors. Here we present an enzymatic, optical glucose sensor element for measurement inside microfluidic systems. The miniaturized glucose sensor (Ø 1 mm) is fabricated together with a reference oxygen sensor onto biocompatible, pressure-sensitive adhesive tape for easy integration inside microfluidic systems. Furthermore, the proposed microfluidic system can be used as plug and play sensor system with existing MPS. It was characterized under cell culture conditions (37 °C and pH 7.4) for five days, exhibiting minor drift (3% day-1). The influence of further cell culture parameters like oxygen concentration, pH, flow rate, and sterilization methods was investigated. The plug-and-play system was used for at-line measurements of glucose levels in (static) cell culture and achieved good agreement with a commercially available glucose sensor. In conclusion, we developed an optical glucose sensor element that can be easily integrated in microfluidic systems and is able to perform stable glucose measurements under cell culture conditions.

Dual-lifetime referencing (DLR): a powerful method for on-line measurement of internal pH in carrier-bound immobilized biocatalysts.

BACKGROUND: Industrial-scale biocatalytic synthesis of fine chemicals occurs preferentially as continuous processes employing immobilized enzymes on insoluble porous carriers. Diffusional effects in these systems often create substrate and product concentration gradients between bulk liquid and the carrier. Moreover, some widely-used biotransformation processes induce changes in proton concentration. Unlike the bulk pH, which is usually controlled at a suitable value, the intraparticle pH of immobilized enzymes may deviate significantly from its activity and stability optima. The magnitude of the resulting pH gradient depends on the ratio of characteristic times for enzymatic reaction and on mass transfer (the latter is strongly influenced by geometrical features of the porous carrier). Design and selection of optimally performing enzyme immobilizates would therefore benefit largely from experimental studies of the intraparticle pH environment. Here, a simple and non-invasive method based on dual-lifetime referencing (DLR) for pH determination in immobilized enzymes is introduced. The technique is applicable to other systems in which particles are kept in suspension by agitation. RESULTS: The DLR method employs fluorescein as pH-sensitive luminophore and Ru(II) tris(4,7-diphenyl-1,10-phenantroline), abbreviated Ru(dpp), as the reference luminophore. Luminescence intensities of the two luminophores are converted into an overall phase shift suitable for pH determination in the range 5.0-8.0. Sepabeads EC-EP were labeled by physically incorporating lipophilic variants of the two luminophores into their polymeric matrix. These beads were employed as carriers for immobilization of cephalosporin C amidase (a model enzyme of industrial relevance). The luminophores did not interfere with the enzyme immobilization characteristics. Analytical intraparticle pH determination was optimized for sensitivity, reproducibility and signal stability under conditions of continuous measurement. During hydrolysis of cephalosporin C by the immobilizate in a stirred reactor with bulk pH maintained at 8.0, the intraparticle pH dropped initially by about 1 pH unit and gradually returned to the bulk pH, reflecting the depletion of substrate from solution. These results support measurement of intraparticle pH as a potential analytical processing tool for proton-forming/consuming biotransformations catalyzed by carrier-bound immobilized enzymes. CONCLUSIONS: Fluorescein and Ru(dpp) constitute a useful pair of luminophores in by DLR-based intraparticle pH monitoring. The pH range accessible by the chosen DLR system overlaps favorably with the pH ranges at which enzymes are optimally active and stable. DLR removes the restriction of working with static immobilized enzyme particles, enabling suspensions of particles to be characterized also. The pH gradient developed between particle and bulk liquid during reaction steady state is an important carrier selection parameter for enzyme immobilization and optimization of biocatalytic conversion processes. Determination of this parameter was rendered possible by the presented DLR method.

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.

Fusing spheroids to aligned µ-tissues in a heart-on-chip featuring oxygen sensing and electrical pacing capabilities.

Over the last decade, Organ-on-Chip (OoC) emerged as a promising technology for advanced in vitro models, recapitulating key physiological cues. OoC approaches tailored for cardiac tissue engineering resulted in a variety of platforms, some of which integrate stimulation or probing capabilities. Due to manual handling processes, however, a large-scale standardized and robust tissue generation, applicable in an industrial setting, is still out of reach. Here, we present a novel cell injection and tissue generation concept relying on spheroids, which can be produced in large quantities and uniform size from induced pluripotent stem cell-derived human cardiomyocytes. Hydrostatic flow transports and accumulates spheroids in dogbone-shaped tissue chambers, which subsequently fuse and form aligned, contracting cardiac muscle fibers. Furthermore, we demonstrate electrical stimulation capabilities by utilizing fluidic media connectors as electrodes and provide the blueprint of a low-cost, open-source, scriptable pulse generator. We report on a novel integration strategy of optical O2 sensor spots into resin-based microfluidic systems, enabling in situ determination of O2 partial pressures. Finally, a proof-of-concept demonstrating electrical stimulation combined with in situ monitoring of metabolic activity in cardiac tissues is provided. The developed system thus opens the door for advanced OoCs integrating biophysical stimulation as well as probing capabilities and serves as a blueprint for the facile and robust generation of high density microtissues in microfluidic modules amenable to scaling-up and automation.