Sanjay Lama's research works | Leipzig University and other places

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Development of an automated platform for monitoring microfluidic reactors through multi-reactor integration and online (chip-)LC/MS-detection

Preprint

February 2024

44 Reads

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This work presents a novel microfluidic screening setup with real-time analytics for investigating reactions with immobilised biocatalysts. The setup combines microreactor technology, multi-reactor integration, and online (chip-)LC/MS analysis in a sequential automated workflow. We utilized in-house manufactured fused-silica glass chips as reusable packed-bed microreactors interconnected as individual tube reactors. The potential of this setup was showcased by conducting and optimising a biocatalytic aromatic bromination reaction as the first proof of concept using immobilised vanadium-dependent haloperoxidase from Curvularia inaequalis (CiVHPO). The fusion of a HaloTagTM to CiVHPO was used for efficient and mild covalent linkage of the enzyme onto chloroalkane-functionalized particles. Then, the biotransformation was continuously monitored with automated LC/MS data acquisition in a data-rich manner. By further developing the automation principle, it was possible to sequentially screen multiple different connected packed-bed microreactors for reaction optimization while using only miniature amounts of reactants and biocatalyst. Finally, we present a fast and modular chipHPLC solution for online analysis to reduce the overall solvent consumption by over 80%. We established a modern microfluidic platform for real-time reaction monitoring and evaluation of biocatalytic reactions through automation of the reactant feed integration, flexible microreactor selection, and online LC/MS analysis.

Fig. 1 schematic sketch of the presented packed-bed multi-microreactor setup with automated reactor selection and on-line (chip)-LC/MS-detection.

Fig. 2 Monitoring the performance of the immobilised CiVHPOHalo enzyme in a packed-bed microreactor by long-term experiments of the bromination model reaction (scale of 1 10 mM) with constant reactant feed and online HPLC/MS-detection. For these runs, no byproduct formation was visible. A) stacked view of all acquired chromatograms (run 1: n=80 chromatograms, approx. 20 h, sampling each 15 min). Three peaks are shown as dominant species, indicating the buffer, reactant 1, and brominated product 2 (EIC only for one brominated product isotope shown). B) Visualisation of the product area fraction in relation to the reactant species over time for three consecutive runs using the same packed-bed microreactor (only one brominated product 2 isotope area considered for visualization). The reactor was flushed overnight with buffer, before introducing a new reaction sample (run 2 & 3: each n=80; each approx. 22 h). Reactor: packed with CiVHPOHalo on ProntoSil particles (∅ 5 µm, loading f = 10.4 µg·mg -1 ), rct. pump: 0.2 µl·min -1 50 mM MES-buffer (residence time approx. 40 s, flushing sample loop with 2 µl·min -1 for 3 min at start; dilution: 2 µl·min -1 MeCN:H 2 O, 60:40 vol% with 50 mM MES as sample); Analysis: Zorbax Eclipse Plus (C18, 4.6x100 mm, 3.5 µm, Agilent), 600 µl·min -1 MeCN:H 2 O (70:30 vol% with 0.1% FA), 60 bar at pump, 2 µl injection volume.

Fig. 3 Automated sequential sample screening approach for monitoring five connected packed-bed microreactors (n=258 chromatograms, approx. 43 h, sampling each 10 min). Each reactor position selected (pos. 2-6) were sampled by the autosampler with a varying reaction mixture (40 µl sample, each reactor run n=16, approx. 160 min; all sample compositions in the ESI in Table S1). For comparison was the last microreactor channel only half-packed (*). Before each reactor run, multiple blank acquisitions were acquired for pump or reactant feed observation and likewise, a washing step was conducted after each reactor run (pos. 1: blank capillary). A) Waterfall diagram of all acquired chromatograms. (EIC only for one brominated product 2 isotope shown) B) Integrated areas of the reactant 1 and product 2 (only one brominated product 2 isotope shown here). The reactant 1 conversion is also shown, calculated by comparing the peak area of the reactant 1 bypassing the reactor before the run with the actual run. Detailed description of the sequence, reaction parameters and information on byproduct S2-S5 formation can be found in the ESI in Section S4.3. Reactor: packed with CiVHPOHalo on ProntoSil particles (∅ 5 µm, loading f = 20.6 µg·mg -1 ), rct. pump: 0.2 µl·min -1 50 mM MES-buffer (residence time approx. 40 s, no dilution); Analysis: Zorbax Eclipse Plus (C18, 4.6x100 mm, 3.5 µm, Agilent), 600 µl·min -1 MeCN:H 2 O (70:30 vol% with 0.1% FA), 51 bar at pump, 0.2 µl injection volume. "w"/"B": washing / blank pump, "r": blank sample, "R1": reactor run.

Fig. 4 Integration of chipHPLC as low solvent consumption alternative to conventional LC/MS in the analytical setup. A) Photograph of the chipHPLC positioned in front of the ESI-source. B) Example of the achieved chipHPLC separation for the model reaction coupled to a packed-bed microreactor (n=25, approx. 2h, sampling each 5 min; EIC only for one brominated product 2 isotope shown). Reactor: packed with CiVHPOHalo on ProntoSil particles (∅ 5 µm), rct. pump: 0.2 µl·min -1 50 mM MES (residence time approx. 40 s, flushing sample loop with 2 µl·min -1 for 3 min at start, dilution: 2 µl·min -1 MeCN:H 2 O, 60:40 vol% with 50 mM MES as sample); Analysis: Xbridge particles 35 mm column length (C18, ∅ 2.5 µm, Agilent), eluent flow: 75 µl·min -1 MeCN:H 2 O (50:50 vol% with 0.1% FA), during elution mode: 72 bar at pump, 70 bar at chip, 5 µl injection volume, 4 s injection time.

Fig. 7 A) Schematic of the instrumental setup for continuous microreactor operation with LC/MS-detection. The integrated selector valves enabled the selection of up to 10 different microreactor positions of the fused-silica glass chip, whereas the first connection was used mostly for an additional blank capillary. B) Variation of the analytical section of the instrumental setup for low consumption chipHPLC integration. Detailed description of the injection principle and capillary length list can be found in the ESI in section S2.

Development of an automated platform for monitoring microfluidic reactors through multi-reactor integration and online (chip-)LC/MS-detection

January 2024

131 Reads

1 Citation

Reaction Chemistry & Engineering

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Sanjay Lama’s research while affiliated with Leipzig University and other places

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