Real‐time pH monitoring of industrially relevant enzymatic reactions in a microfluidic side‐entry reactor (μSER) shows potential for pH control

Abstract

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 ...

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