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rsc.li/reaction-engineering
Linking fundamental chemistry and engineering to create scalable, efficient processes
Reaction Chemistry
& Engineering
rsc.li/reaction-engineering
ISSN 2058-9883
PAPER
Timothy Noel et al.
A convenient numbering-up strategy for the scale-up of gas-
liquid photoredox catalysis in flow
Volume 1
Number 1
1 February 2016
Pages 1-120
Linking fundamental chemistry and engineering to create scalable, efficient processes
Reaction Chemistry
& Engineering
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Schmidt, S. Lama, M. Polack, C. Weise, T. Oestereich, R. Warias, T. Gulder and D. Belder, React. Chem.
Eng., 2024, DOI: 10.1039/D4RE00004H.
ARTICLE
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Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Development of an automated platform for monitoring
microfluidic reactors through multi-reactor integration and online
(chip-)LC/MS-detection
Hannes Westphal,a† Simon Schmidt,b† Sanjay Lama,a Matthias Polack,a Chris Weise,a Toni
Oestereich,a Rico Warias,a Tanja Gulder,*b,c,d Detlev Belder*a
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.
Introduction
The efficient utilisation of biocatalysis plays a pivotal role in
achieving the objective of implementing environmentally-
friendly chemistry in industrial processes.1,2 Enzymes enable
highly selective reactions under mild and economically friendly
conditions, leading to their increasing commercial use, including
the synthesis of valuable fine chemicals and small-molecule
active pharmaceutical ingredients (APIs).3,4
The remarkable surge in enzyme-based processes can be
attributed to advances in biotechnology and enzyme
engineering,5,6 such as advanced DNA sequencing of genomes,
directed evolution, especially in combination with genetic code
expansion, and computer-assisted methods.7,8 These
developments have facilitated the discovery and development
of new, more robust, and powerful enzymes which can also be
optimised for non-natural substrates, thus expanding
biocatalytic methodologies. Some researchers even consider
this period to be the "Golden Age of Biocatalysis".9
These novel enzymes can be made even more robust by
immobilisation,10–12 for which a wide selection of solid support
materials and linking methods are available.13,14 Next to
considerable cost reduction through increased material
stability, a more comprehensive range of reaction conditions
can be used in water-immiscible organic solvents.15
Furthermore, enzyme immobilisation not only simplifies the
handling of the biocatalysts and the separation of the product
from it, but also enables the transition of biocatalytic processes
into continuous flow systems as immobilised enzyme reactors
(IMERs).16–20 Various conventional continuous flow
technologies have already been developed and are
commercially available,21–24 offering significant benefits,
including process simplification, improved and consistent
product quality, easier downstream processing, and the
integration of online analytics.
Biocatalysis can benefit significantly from using smaller reaction
spaces, particularly in microreactors. The literature shows an
increasing number of biotransformations in the microflow
regime,25,26 often utilising so-called microfluidic immobilised
a.H. Westphal, S. Lama, M. Polack, C. Weise, T. Oestereich, R. Warias, D. Belder,
Institute of Analytical Chemistry, Leipzig University, Linnéstraße 3, 04103 Leipzig,
Germany, E-mail: belder@uni-leipzig.de
b.S. Schmidt, T. Gulder, Institute of Organic Chemistry, Faculty of Chemistry and
Mineralogy, Leipzig University, Johannisallee 29, 04103 Leipzig, Germany, E-mail:
tanja.gulder@uni-leipzig.de
c. T. Gulder, Organic Chemistry I, Saarland University, 66123 Saarbruecken,
Germany
d.T. Gulder, Synthesis of Natural-Product Derived Drugs, Helmholtz Institute for
Pharmaceutical Research Saarland (HIPS) Helmholtz Centre for Infection Research
(HZI), 66123 Saarbrücken, Germany
† These authors contributed equally to this manuscript.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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enzyme reactors (μ-IMERs).26–30 For both IMERs and their
microfluidic versions, a range of online analytical methods,
including chromatographic and spectroscopic techniques,31–34
were tested in combination with these systems for
comprehensive evaluation of the reaction outcome and
performance.
Downsizing of chemical flow reactions has, in general, several
advantages. This reduces the consumption of reactants such as
high-value APIs, immobilised catalysts, and expensive cofactors
and improves heat and mass transfer, real-time process control,
and mixing behaviour in a shorter time and spatial
dimension.35,36 This approach allows processes to be conducted
more safely and reduces waste generation. The enhanced flow
control in continuous microfluidic systems also facilitates
automation and parallelisation,37,38 leading to the development
of several low-scale synthesizers.39,40
The field of reactor technology is rapidly advancing, driven by
developments in data processing, which encompass the
utilisation of recently matured data science disciplines,41–44
machine learning,45,46 and sophisticated artificial intelligence
methods.47–49 This inflicts various discussed research areas such
as microfluidics,50–52 enzyme development,53–57 or reaction
optimisation.58–60 The implementation of these methods
underlines the growing necessity for reliable, rapid raw data
generation and instrumental hardware control, preferably
within continuous flow systems integrated with real-time online
analysis.61
For biocatalysis, however, there currently needs to be
automated and data-rich methods for rapid and reliable
validation of immobilised enzymes and screening of
biotransformations.62 Consequently, tedious manual reaction
screening is often required. Microfluidic devices are particularly
suited for this purpose due to their low consumption, precise
fluidic control, and versatility for their integration as either
highly integrated or modular lab-on-a-chip devices.63–65
To address this challenge, we propose a biocatalytic automated
screening platform in microflow coupled with online LC/MS
detection. We recently investigated biotransformations in a
digital microfluidic (DMF) approach.66 Building on our previous
work with various integrated microfluidic devices for studying
immobilised organocatalysts,67–69 we have now transitioned
these biotransformations into a microflow platform, leading to
improved automation, reactor control, and expanded
applications in biocatalytic processes.
We used the vanadium-dependent haloperoxidase (CiVHPO)
from the fungus Curvularia inaequalis as a model enzyme for
evaluating a biocatalytic bromination reaction.70–72 The enzyme
exhibits remarkable properties, including high stability to heat
and organic solvents, a broad substrate scope, and the ability to
use H2O2 as an easily accessible cosubstrate.73 As a result,
numerous papers have been published in recent years utilizing
the efficient introduction of halogen atoms by CiVHPO in a wide
range of organic compounds.74–80 We decided to covalently
attach the CiVHPO to the solid support for better handling.
Therefore, we genetically added the HaloTagTM to our protein
of interest (CiVHPOHalo) and then linked it via a chloroalkane to
ProntoSIL particles. The HaloTagTM method is highly selective
and oriented, enabling reproducibility and easy adaptation to
other biocatalysts.81–84
After immobilisation, the enzyme was packed into a custom-
made microfluidic fused-silica glass chip and was evaluated
using the presented system. For screening applications, this
setup has been designed to sequentially address multiple
parallel packed-bed microreactors with different content or
reactants in an automated workflow that allows for high
variability. The approach minimises the reactant and catalyst
consumption, emphasising the efficiency of this screening
platform with minimal laboratory effort. We integrated online
analysis to enable continuous, long-term monitoring by
coupling the system with LC/MS and implementing automated
injections. Additionally, we propose miniaturizing the analytical
method using chipHPLC, leveraging our previous expertise in
this area,85–88 to significantly reduce solvent consumption in the
analytical section and accelerate the separation time.
Fig. 1 schematic sketch of the presented packed-bed multi-microreactor setup with automated reactor selection and on-line (chip)-LC/MS-detection.
Page 2 of 10Reaction Chemistry & Engineering
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Results and Discussion
Long-term studies for immobilised enzyme evaluation
We first immobilised the CiHVPO enzyme using the HaloTagTM
immobilisation technique as a highly selective and oriented
one-step covalent linking method under mild conditions. Having
sorted out a reproducible covalent linking of our enzyme to
silica particles, we packed the material inside a packed-bed
microreactor and evaluated it in continuous flow using an
aromatic bromination of pyrroles as model biotransformation.
Various runs are presented below to illustrate the application of
the setup for continuous flow microreactor sequencing. These
measurements were conducted to establish the following
points: First, to confirm stable enzyme activity over several
days; second, to demonstrate that no enzyme leakage is
observed with covalent HaloTagTM immobilisation; and third, to
validate stable and reproducible reaction conversion for our
model biotransformation. To achieve this, the microreactor was
monitored for approx. 22 h, chromatograms were measured
every 15 min (Fig. 2 A), and a constant reactant stream was
applied. Two consecutive runs of the same microreactor were
then carried out as recycling experiments, with the reactor
washed with buffer overnight after each run (Fig. 2 B) and the
fraction of the product 2 signal visualised (for relative
evaluation, only one brominated product isotope considered).
These long-term monitoring runs of the immobilised enzyme
successfully demonstrated good operational stability with only
a slight drop in biocatalytic activity over time. Interestingly,
some enzyme activity was regained during the overnight
washing step.
To confirm the selective immobilisation only via the HaloTagTM,
measurements were conducted with particles without
chloroalkane linker. In this case, the enzyme was presumably
attached to the ProntoSIL particles through non-covalent
interactions during the enzyme immobilisation process. These
interactions, however, proved insufficient to withstand the flow
conditions, resulting in a significant loss of activity due to
clearance of the enzyme from the reactor over time (Fig. 2 B).
Similarly, blank ProntoSil particles with a linker but without
enzyme addition showed no conversion at all. Additional
information and further runs at different reaction conditions
can be found in the ESI at section S5.1.
Multi-reactor screening approach
Two multi-selector valves were integrated into the setup to
increase control for reaction monitoring or screening purposes,
allowing for switching between several different microreactor
positions during individual pre-programmed runs. This
configuration enables easy integration of multiple packed-bed
microreactors in parallel. In addition, a blank capillary was
added at the first position, which facilitates acquiring blank
measurements of the reactant feed or sole pump feed while
bypassing the reactor. Furthermore, by switching off the
reactant feed, reactor positions can be flushed between runs,
and the efficiency of the washing process can be monitored. An
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:H2O, 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:H2O (70:30 vol% with 0.1% FA), 60 bar at pump, 2 µl injection volume.
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initial validation run using a single connected packed-bed
microreactor and a blank capillary is described in the ESI in
Section S5.2. A constant reactant feed was provided for that run
and then directed to the two reactor positions. Monitoring the
signals during reactor switching, the flow behaviour was
investigated and validated that no carry-over effects were
present.
To further enhance the flexibility of the reactant feed, an
autosampler was integrated as an automated tool for reactant
feed variation, allowing for sequential screening of various
manually prepared samples in an automated workflow. Due to
the low reactor flow rate, the autosampler loop provided a
constant reactant feed for multiple hours (40 µl, 0.2 µl/min,
approx. 160 min reactant plug plateau). Thus, the platform can
be used for testing different reaction conditions, which,
combined with the selector valves, can be addressed to multiple
different microreactors while minimising enzyme and reactant
consumption. This functionality was demonstrated by
sequentially testing five adjacent freshly packed reactor
channels with different reaction mixtures in a single automated
run, as shown in Fig. 3 (all channels packed with CiVHPOHalo
immobilised particles; last reactor only half-packed for
comparison; sampling each 10 min, with each reactor run
approx. 160 min and n=16 chromatograms; in total n=258
chromatograms, approx. 43 h).
Blank measurements bypassing the reactor were performed
before each separate run to observe the pump or reactant feed
background before injecting the sample into the reactor. Those
measurements could directly be used to calculate the
reactant 1 conversion in the subsequent reactor runs (Fig. 3 B).
Additionally, washing steps were conducted after each run to
flush the respective reactant.
As proof of principle, the effect of varying reactant 1
concentrations of hydrogen peroxide, sodium vanadate, and
ammonium bromide on the examined biocatalytic bromination
reaction was screened (detailed reactant compositions in the
ESI at Table S1). A higher hydrogen peroxide proportion (R2:
1.75 eq. H2O2 and 2 eq. NH4Br instead of initial R1: 1.0 eq. and
7.0 eq. mM, respectively) resulted in a higher conversion and
product 2 area fraction. However, this led to byproduct
formation, which was assigned as an oxygenated species S2-S5
(not shown here; discussion in ESI section S4.3).
Further increasing the vanadium cofactor (R3: 2 eq. Na3VO4
instead of initial R2: 1 eq.) or the ACN fraction (R4: 70% ACN
instead of initial R2: 40%) led to less total reactant conversion,
which can be attributed to reduced byproduct S2-S5 formation.
At the same time, the product 2 signal remained consistently
high. As expected, the last microreactor, containing only a half-
packed column (R5), resulted in less total conversion and
product 2 formation.
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:H2O (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.
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Overall, the automated multi-reactor screening platform
demonstrated the capability for efficient and rapid screening of
various reaction samples with different compositions, allowing
for fast data generation and optimisation of biocatalytic
transformations or as validation feedback for enzyme
optimization processes (automation and sequencing details, as
well as additional experimental details, can be withdrawn from
the ESI at section S5.2).
Integration of chipHPLC analysis
As demonstrated above, our microreactor setup allows for long-
time measurements with low consumption of reactants and
solvents and the reusability of our biocatalyst, making it an
efficient and sustainable method. However, compared to the
microreactor, using a commercially available C18-column in the
online analytic setup still involves relatively high solvent
consumption (600 µl·min-1 or approx. 864 ml·d-1). To further
reduce solvent usage, we integrated a chipHPLC-setup as a
modular alternative to the commercially used C18-column
(Fig. 4 A; the simplified setup is based on a recent joint
publication88). Initially, the chipHPLC-setup was integrated, the
injection principle visualised, and the duty cycle was automated
similarly (detailed description in the ESI in section S3). The
separation was optimized by sampling the reaction performed
in batch through a syringe as sample feed. Subsequently, the
chipHPLC was coupled to a packed-bed microreactor, as
presented above. The achieved separation is presented in
Fig. 4 B. Herein, the chipHPLC system offered a significantly
faster separation time below 2 min, leading to an increased
acquisition frequency and, thus, faster real-time monitoring. In
addition, such short acquisition times could be used in the
future, for instance, in kinetic studies of the initial phase of a
catalysed reaction. The system also demonstrated good
operational stability with minimal solvent consumption down to
75 µl·min-1 or approx. 108 ml·d-1, resulting in a reduction of
>80%.
The rapid analysis time with minimal consumption
demonstrates this modular approach as optimal for analytical
setup integration. Further information on the injection principle
and conducted chipHPLC runs, including pressure data, a long-
term stability test, and a detailed comparison of the different
methods, can be found in the ESI in section S5.3.
Methods
Microfluidic devices and preparational steps
Microreactor-chip: The fused-silica glass chips used in this study
as packed-bed microreactors were manufactured by an in-
house method, using a selective laser-induced etching (SLE)
process, wafer-to-wafer alignment, and direct glass-glass
bonding, followed by a high-temperature fusion bonding step
of two structured wafers with subsequent chip dicing. The chip
layout was created using CAD-software Autodesk Inventor
Professional 2019 (San Rafael, CA, USA) and converted with
CAM-software (Alphacam 2017 R2, Vero Software GmbH, Neu-
Isenburg, DE) into a corresponding toolpath for the SLE-device
(FEMTOprint f200 aHead P2, Muzzano, CHE). An ultrashort
pulsed IR-laser (1030 nm, 400 fs, 230 nJ) was employed to
structure the design onto a 4-inch fused silica wafer (thickness
1 mm), followed by a wet chemical-etching step in hot
potassium hydroxide solution (8 M, 85 °C, 6 h). A detailed
description of the manufacturing process can be found in the
ESI in section S1.1.
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:H2O, 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:H2O (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.
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As shown in Fig. 5 A, the chip design consists of multiple
adjacent reactor channels located directly after the chip inlets.
Each channel features a semi-circular reactor-bed (measured 76
x 26 x 2 mm; e.g., 15 reactor channels, each with 19 mm length,
150 µm width, and 65 µm depth, resulting in an empty reactor
volume of approx. 265 nl and 132 nl packed). Each reactor
channel can be separately filled with particulate material and
terminates in an integrated weir-structure or µfrit, which
retains the packed particles via the keystone effect (similar as
we previously presented,89 µfrit shown in Fig. 5 C, µfrit channels
measuring 150 µm in length, 15-20 µm in width and depth).
Beyond the weir-structure, there is an additional short channel
(0.9 mm) before reaching the chip outlet. The conical inlet and
outlet holes of the channels can be connected by utilising a
custom-built high-pressure steel clamp system, allowing for a
world-to-chip connection.90 The prepared packed-bed flow
reactor can then be directly integrated into the fluidic setup by
connection to the reactant feed. For reusability, the weir-
structure is positioned on only one side of the chip, enabling
reactor operation in a single direction and the possibility of
subsequent reactor recycling by flushing the reactor in the
opposite direction. This allowed for efficient removal of the
particulate material and facilitated the repacking of the reactor
with different materials.
LC-chip: For the final integrated chip-based separations, a
bonded borosilicate glass chip was utilised, which was
manufactured externally according to an in-house protocol (by
iX-factory GmbH, now part of Micronit GmbH, Dortmund,
Germany), including conventional photolithography, wet
etching, and subsequent high-temperature fusion bonding (a
process also described elsewhere,86,90–92 chip dimensions: 45
x 10 x 2.2 mm). In general, the chip design, as shown in Fig. 6,
consists of an integrated injection-cross connected to a slurry-
packed chromatographic column located in a separation
channel with a semicircular cross-section (35 mm length, 90 µm
max width, 40 µm depth). At both channel ends,
photopolymerised frits were integrated as particle-retaining
structures. As stationary phase material, either XBridge (C18,
∅ 2.5 µm, Waters, Milford, USA) or Poroshell particles (C18,
∅ 2.7 µm, Agilent, Santa Clara, USA) were used and packed into
the separation column through an additional packing channel.
The packing channel was then sealed with a photopolymerised
plug, and a monolithic pyramidal electrospray emitter was
ground and hydrophobised before use. Similar to the µreactor
chip, all fluidic connections were realised by high-pressure
clamps as described above.90 More details of the preparation
steps can be found in the ESI in section S1.2.
Slurry-packing: In both chip variants, the particles were
integrated by slurry-packing (either for the biocatalytic material
or as a separation column). The chip was positioned in an
ultrasonic bath and connected to an HPLC-pump and 6-port-
valve with a slurry-filled sample loop (approx. 2-3 mg/ml
particle slurry). The process could be reversed for the reactor
chip by flushing the reactor from the opposite direction.
Fluidic Circuit and instrumental setup
The following describes the fluidic instrumental setup, including
all components and connections. The setup is shown
schematically in Fig. 7 A and can be divided into one section for
continuous microreactor operation and another section for
online LC/MS-analysis, which can be performed by either a
conventional or a chipHPLC approach.
All system flows were generated using three HPLC pumps in
combination. The first pump provided the sample stream
through the microreactor at a relatively low flowrate
(0.2 µl/min, 40:60 ACN:H2O (v/v) + 50 mM MES-buffer pH 6.0;
Fig. 5 Fused-silica glass chip used as packed-bed microreactors.
A) photograph of a fully manufactured chip with adjacent 15 microreactor
channels. B) Microscopic picture of the integrated µfrit as particle retaining
structure, with an already packed channel on the left side. C) Dimensions of
the µfrit by laser-scanning microscopic measurements (detailed information
can be found in the ESI section S1.1).
Fig. 6 Borosilicate glass chipHPLC with integrated injection cross, packed
separation column, and monolithic electrospray emitter (as we presented
before). A) photograph of a fully manufactured chip. B) Schematic sketch of
the chipHPLC describing all inlets and modifications for integration of the
separation column (detailed information can be found in the ESI section S1.2).
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LC-20AD, Shimadzu, Kyoto, Japan). To provide the respective
reactant feed, either a sample loop was connected to an
additional valve (e.g., 0.3-1 ml sample reservoir for long-term
monitoring experiments; refillable via an external PEEK needle
port adapter) or an autosampler to facilitate sample variation
during individual runs (40 µl loop, G1377A, Agilent). In certain
runs, a second pump was used for sample dilution past the
microreactor positions before online analysis (1:10 dilution,
1260 Infinity isocratic HPLC pump, Agilent). The final pump was
utilised in the analytical section to generate the eluent flow for
LC/MS-analysis using a binary pump (600 µl/min, 70:30
ACN:H2O (v/v); 1260 Infinity binary HPLC pump, Agilent). The
fluidic circuit was realised by combining multiple Nanovolume
valves (either two-position or selector valves, with 360 µm
connections, Cheminert, VICI AG, Schenkon, Switzerland),
which were connected through commercially available PEEK
and FS capillary tubing (mostly 360 µm OD, 50-100 µm ID, VICI
AG) and high-pressure PEEK fittings on a custom metal stage.
The microreactors, connected between the two selector valves,
can be selected from ten different positions by switching the
fluidic path of the two valves for increased flexibility during
automated reaction monitoring.
For online analysis, the reactor stream was guided through an
additional 2-position valve (either 0.2 or 2 µl injection loop,
depending on whether a reactant sample feed dilution was
used), which could be sequentially sampled onto a
chromatographic column (Zorbax Eclipse Plus C18, 4.6x100 mm,
3.5 µm, Agilent) and detected by an ESI-MS-system (AmaZon SL,
Bruker Daltonics GmbH, Bremen, Germany).
Electric actuators controlled all valves, while the operation was
automated by a Clarity chromatography data station combined
with a Colibrick A/D converter box (DataApex, Prague, Czech
Republic). These modules enhanced pump control and pressure
monitoring and enabled automated injection sequences of the
reactor effluent for LC/MS-detection. This included sampling
every 10-15 min and starting acquisitions by connecting to the
auxiliary ports of the mass spectrometer. In addition, it was
possible to sequentially address multiple microreactor positions
in single runs with individual reactant sampling by controlling
the selector valves and the connected autosampler. Further
information about the instrumental setup and a detailed
description of the automation principle, auxiliary connections,
and sequencing for multi-reactor operation can be found in the
ESI in section S2 & S3.
ChipHPLC integration to the instrumental setup
A modified analytical section was later employed to enable
chip-based separations as a fast but low-consumption
alternative to the commercially used C18-column (the general
chipHPLC-setup was based on a recent joint publication88). For
that purpose, the fluidic connections of the setup were
modified according to Fig. 7 B, and the custom metal stage was
connected directly to the MS source. The chip was mounted on
an xyz-linear translation stage (T12XYZ, Thorlabs GmbH,
Dachau, Germany), and the chip emitter was precisely
positioned in front of the ESI-inlet. Furthermore, the flow rate
of the binary eluent pump was lowered accordingly. Initially, the
injection principle was evaluated and visualised by a sampling
of fluorescent dyes. Then, the duty cycle of the injection
principle was similarly automated as before (more details on
the setup can be found in the ESI in section S2.2 and for the
injection principle in S5.3).
Immobilisation strategy and model reaction
Enzyme immobilisation: Typically, 10 mg NH2-coated ProntoSIL
was suspended in ethanol (1 ml) followed by addition of the
HaloTagTM-targeting chloroalkane linker (S1, for structure, see
ESI Fig. S14 (104 µl, 100 mM stock solution in ethanol) and
trimethylamine (5.0 µl). After incubation at rt for 16 h at 1200
rpm shaking, the particles were centrifuged and washed with
ethanol (3 × 1 ml). Linker loading was determined afterward by
Kaiser Test (for detailed information, see ESI section S4.2).93 For
reactor runs using particles without a linker the same strategy
was used. However, only ethanol was added instead of the
HaloTagTM-targeting chloroalkane linker S1 solution. Finally,
CiVHPOHalo (15.9 µl, 3.142 µg/µl stock solution) and buffer (484
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.
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µl, containing 50 mM Tris pH 6.0 and 100 µM Na3VO4) were
added to the particles, and the mixture was incubated at rt for
1 h at 1000 rpm shaking. The particles were washed with MES
buffer (3 × 1 ml). Interestingly, after immobilisation, a good
retention of 68% of the initial activity for CiVHPOHalo on
ProntoSIL was observed (for detailed information, see ESI
Fig. S20).
Enzyme quantification: The enzyme loading on the solid support
was determined by quantifying the non-bound enzyme
concentrations of CiVHPOHalo in the supernatants using the
monochlorodimedon (MCD) Assay (for detailed information,
see ESI Fig. S19) after the immobilisation protocol.94 By
measuring the decrease of MCD absorbance (290 nm) over
time, enzyme concentrations could be calculated using a
calibration slope. With this information, the amount of enzyme
on the particles was determined. Usually, 7.47 – 20.6 µg
CiVHPOHalo was immobilised per 1 mg of chloroalkane-
modified particle. CiVHPOHalo was also immobilised on the
amine functionalised ProntoSIL particles by non-covalent
interactions (up to 12.2 – 18.8 µg of CiVHPOHalo was
immobilised per 1 mg of amine particle).
Biocatalytic transformations: As a model reaction for the
presented integrated system, the bromination of ethyl-3,5-
dimethyl pyrrole-2-carboxylate (1, 10 mM) to ethyl-4-bromo-
3,5-dimethyl pyrrole-2-carboxylate (2) was investigated as
shown in Fig. 2 A. The standard reaction mixture consisted of
MES-buffer (pH 6.0, 50 mM) in 40:60 ACN:H2O (v/v), with NH4Br
as bromination source (70 mM), Na3VO4 as cofactor (1 mM),
and H2O2 as oxidation agent (10 mM). A detailed overview of
further tested compositions and potential side reactions can be
found in the ESI in section S4.3.
Conclusions
In summary, our presented chip-based microfluidic setup offers
a robust platform for monitoring biocatalytic transformations in
packed-bed microreactors, featuring an automated injection
workflow with almost real-time online LC/MS detection.
Furthermore, we integrated a modular chipHPLC-based unit,
which serves as a low-consumption alternative to the
conventional C18-column used initially and significantly
accelerates the separation process, while reducing solvent
consumption by over 80%.
Our investigation focused on immobilising a vanadium-
dependent haloperoxidase onto silica particles and packing
them into microfluidic fused-silica chips. The performance of
our newly established system was evaluated by employing a
biocatalytic bromination as a model reaction. The covalent
immobilisation strategy showed good stability while
maintaining high enzyme activity during long-term monitoring
experiments using only minute amounts of catalytic material (1-
10 mg range).
The setup allows for the sequential monitoring of multiple
interconnected microreactors by external valve control through
reactor position switching. We showcased this capability by
testing various sample compositions of the model
biotransformation across multiple continuous microreactors in
a single automated run, including blank measurements
bypassing the microreactor.
The versatility of this approach allows for easy adaption to a
parallel screening of multiple reactions or different immobilised
catalytic materials in separate microreactors, making it suitable
for various applications, including data feedback of optimised
enzymes by directed evolution. Due to its deficient substrate
consumption, the system is also well suited for studying APIs
and derivatisations. By combining microfluidic approaches with
extended setup automation and Python-based data evaluation,
such data-rich systems could pave the way for more accessible
connections to upcoming data science disciplines for more
sustainable and efficient chemical processes.
Author Contributions
Conceptualization: H.W., S.S., R.W., S.L., T.G., D.B.;
Investigation: H.W., S.S., S.L., T.O., R.W.; Methodology: H.W.,
S.S., S.L, M.P., R.W., C.W.; Software: H.W.; Writing (original
draft): H.W., S.S., T.G., D.B.; Review & Editing: all authors;
Supervision, Funding, Project administration: T.G., D.B.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank Dr. Daniel Splith (Prof. Grundmann group, Leipzig
University) for assisting with the laser-scanning microscope
measurements. In addition, we would like to thank Monika
Hahn (Prof. Grundmann group, Leipzig University) for assisting
with the wafer dicing. TG and SS thank the Emmy-Noether
program of the German Research Foundation (DFG, GU 1134/3)
for generous funding.
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DOI: 10.1039/D4RE00004H
