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 BF-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 pK 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.

Oxygen plays a fundamental role in cellular energy metabolism, differentiation and cell biology in general. Consequently, in vitro oxygen sensing can be used to assess cell vitality and detect specific mechanisms of toxicity. In 2D in vitro models currently used, the oxygen supply provided by diffusion is generally too low, especially for cells having a high oxygen demand. In organ-on-chip systems, a more physiologic oxygen supply can be generated by establishing unidirectional perfusion. We established oxygen sensors in an easy-to-use and parallelized organ-on-chip system. We demonstrated the applicability of this system by analyzing the influence of fructose (40 mM, 80 mM), ammonium chloride (100 mM) and Na-diclofenac (50 µM, 150 µM, 450 µM, 1500 µM) on primary human hepatocytes (PHH). Fructose treatment for two hours showed an immediate drop of oxygen consumption (OC) with subsequent increase to nearly initial levels. Treatment with 80 mM glucose, 20 mM lactate or 20 mM glycerol did not result in any changes in OC which demonstrates a specific effect of fructose. Application of ammonium chloride for two hours did not show any immediate effects on OC, but qualitatively changed the cellular response to FCCP treatment. Na-diclofenac treatment for 24 hours led to a decrease of the maximal respiration and reserve capacity. We also demonstrated the stability of our system by repeatedly treating cells with 40 mM fructose, which led to similar cell responses on the same day as well as on subsequent days. In conclusion, our system enables in depth analysis of cellular respiration after substrate treatment in an unidirectional perfused organ-on-chip system.

Microbioreactors (MBRs) with a volume below 1 mL are promising alternatives to established cultivation platforms such as shake flasks, lab-scale bioreactors and microtiter plates. Their main advantages are simple automatization and parallelization and the saving of expensive media components and test substances. These advantages are particularly pronounced in small-scale MBRs with a volume below 10 µL. However, most described small-scale MBRs are lacking in process information from integrated sensors due to limited space and sensor technology. Therefore, a novel capillary-wave microbioreactor (cwMBR) with a volume of only 7 µL has the potential to close this gap, as it combines a small volume with integrated sensors for biomass, pH, dissolved oxygen (DO) and glucose concentration. In the cwMBR, pH and DO are measured by established luminescent optical sensors on the bottom of the cwMBR. The novel glucose sensor is based on a modified oxygen sensor, which measures the oxygen uptake of glucose oxidase (GOx) in the presence of glucose up to a concentration of 15 mM. Furthermore, absorbance measurement allows biomass determination. The optical sensors enabled the characterization of an Escherichia coli batch cultivation over 8 h in the cwMBR as proof of concept for further bioprocesses. Hence, the cwMBR with integrated optical sensors has the potential for a wide range of microscale bioprocesses, including cell-based assays, screening applications and process development.