The quantification of key variables such as oxygen, pH, carbon dioxide, glucose, and temperature provides essential information for biological and biotechnological applications and their development. Microfluidic devices offer an opportunity to accelerate research and development in these areas due to their small scale, and the fine control over the microenvironment, provided that these key variables can be measured. Optical sensors are well-suited for this task. They offer non-invasive and non-destructive monitoring of the mentioned variables, and the establishment of time-course profiles without the need for sampling from the microfluidic devices. They can also be implemented in larger systems, facilitating cross-scale comparison of analytical data. This tutorial review presents an overview of the optical sensors and their technology, with a view to support current and potential new users in microfluidics and biotechnology in the implementation of such sensors. It introduces the benefits and challenges of sensor integration, including, their application for microbioreactors. Sensor formats, integration methods, device bonding options, and monitoring options are explained. Luminescent sensors for oxygen, pH, carbon dioxide, glucose and temperature are showcased. Areas where further development is needed are highlighted with the intent to guide future development efforts towards analytes for which reliable, stable, or easily integrated detection methods are not yet available.

The continuous production of high value or difficult to synthesize products is of increasing interest to the pharmaceutical industry. Cascading reaction systems have already been employed for chemical synthesis with great success, allowing a quick change in reaction conditions and addition of new reactants as well as removal of side products. A cascading system can remove the need for isolating unstable intermediates, increasing the yield of a synthetic pathway. Based on the success for chemical synthesis, the question arises how cascading systems could be beneficial to chemo‐enzymatic or biocatalytic synthesis. Microreactors, with their rapid mass and heat transfer, small reaction volumes and short diffusion pathways, are promising tools for the development of such processes. In this mini‐review, the authors provide an overview of recent examples of cascaded microreactors. Special attention will be paid to how microreactors are combined and the challenges as well as opportunities that arise from such combinations. Selected chemical reaction cascades will be used to illustrate this concept, before the discussion is widened to include chemo‐enzymatic and multi‐enzyme cascades. The authors also present the state of the art of online and at‐line monitoring for enzymatic microreactor cascades. Finally, the authors review work‐up and purification steps and their integration with microreactor cascades, highlighting the potential and the challenges of integrated cascades.

Rapid biocatalytic process development and intensification continues to be challenging with currently available methods. Chiral amino‐alcohols are of particular interest as they represent key industrial synthons for the production of complex molecules and optically pure pharmaceuticals. (2S,3R)‐2‐amino‐1,3,4‐butanetriol (ABT), a building block for the synthesis of protease inhibitors and detoxifying agents, can be synthesized from simple, non‐chiral starting materials, by coupling a transketolase‐ and a transaminase‐catalyzed reaction. However, until today, full conversion has not been shown and, typically, long reaction times are reported, making process modifications and improvement challenging. In this contribution, we present a novel microreactor‐based approach based on free enzymes, and we report for the first time full conversion of ABT in a coupled enzyme cascade for both batch and continuous‐flow systems. Using the compartmentalization of the reactions afforded by the microreactor cascade, we overcame inhibitory effects, increased the activity per unit volume, and optimized individual reaction conditions. The transketolase‐catalyzed reaction was completed in under 10 min with a volumetric activity of 3.25 U ml−1. Following optimization of the transaminase‐catalyzed reaction, a volumetric activity of 10.8 U ml−1 was attained which led to full conversion of the coupled reaction in 2 hr. The presented approach illustrates how continuous‐flow microreactors can be applied for the design and optimization of biocatalytic processes.

Monitoring and control of pH is essential for the control of reaction conditions and reaction progress for any biocatalytic or biotechnological process. Microfluidic enzymatic reactors are increasingly proposed for process development, however typically lack instrumentation, such as pH monitoring. We present a microfluidic side-entry reactor (µSER) and demonstrate for the first time real-time pH monitoring of the progression of an enzymatic reaction in a microfluidic reactor as a first step towards achieving pH control. Two different types of optical pH sensors were integrated at several positions in the reactor channel which enabled pH monitoring between pH 3.5 and pH 8.5, thus a broader range than typically reported. The sensors withstood the thermal bonding temperatures typical of microfluidic device fabrication. Additionally, fluidic inputs along the reaction channel were implemented to adjust the pH of the reaction. Time-course profiles of pH were recorded for a transketolase- and a penicillin G acylase-catalyzed reaction. Without pH adjustment, the former showed a pH increase of one pH unit and the latter a pH decrease of about 2.5 pH units. With pH adjustment, the pH drop of the penicillin G acylase-catalyzed reaction was significantly attenuated, the reaction condition kept at a pH suitable for the operation of the enzyme, and the product yield increased. This contribution represents a further step towards fully instrumented and controlled microfluidic reactors for biocatalytic process development. Keywords: Microreactor · Online monitoring · Optical sensor · Penicillin G acylase · pH sensor · TransketolaseCorrespondence: Prof. Nicolas Szita, University College London, Biochemical Engineering, Bernard Katz Building, Gordon Street, London, WC1H 0AH, UK E-mail: n.szita@ucl.ac.uk Abbreviations: 6-APA, 6-amino benzyl penicillanic acid; ERY, L-erythrulose; GA, glycol aldehyde; GC, gas chromatography; HPA, hydroxyl pyruvate; HPLC, high performance liquid chromatography; ISFET, ion-sensitive field effect transistor; PG, penicillin G; PGA, penicillin G acylase; TFA, trifluoroacetic acid; TK, transketolase; µSER, microfluidic side-entry reactor Biotechnol. J. 2017, 12, 1600475 electrode becomes less practical. This integration challenge is exacerbated in microfluidic devices, where channels are normally small, narrow, and enclosed. Reactions can in principle be monitored at-line or off-line, for example with a HPLC or a GC; however, in reactions where a change in a common analyte such as oxygen or pH occurs, online monitoring is preferable, as it provides a direct, i.e. real time, measure of the progress of the reaction. On-line monitoring at the microfluidic scale thus enables the rapid analysis of enzymatic reactions with small amounts of enzymes, and -due to the fine control over the fluid flow afforded by microfluidics -with precise control over reaction conditions. Monitoring of pH can be accomplished using electrochemical sensors, such as ion-sensitive field effect transistors (ISFETs) [3]. Since their ...

Resistivity of optical carbon dioxide chemosensors towards poisoning by acidic gases is significantly improved by using an additional perfluorinated polymer coating making the sensors suitable for long-term measurements.