An automated and compartmented fluidic reactor device for multi-step sample-to-answer processes using magnetic particles

Hübner, Jonas; Heinzler, Raphael; Arlt, Carsten-Rene; Hohmann, Siegfried; Brenner‐Weiß, Gerald; Franzreb, Matthias

doi:10.1039/c6re00219f

Cited by 4 publications

(7 citation statements)

References 43 publications

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“…To characterize the activity of the MEB in the microreactor system with regard to pH, temperature, and loading, MEB were pumped into the reaction tubing of the system. The MEB were separated from the storage buffer (as described before), resuspended in 100 μL of a preheated reaction buffer compartment and pumped to the temperature control module (TCM). Details of the IR‐unit for preheating and the TCM are discussed by Heinzler et al., describing the reactor system.…”

Section: Methodsmentioning

confidence: 99%

“…The MEB were separated from the storage buffer (as described before), [22] resuspended in 100 μL of a preheated reaction buffer compartment and pumped to the temperature control module (TCM). The MEB were separated from the storage buffer (as described before), [22] resuspended in 100 μL of a preheated reaction buffer compartment and pumped to the temperature control module (TCM).…”

Section: Activity Assay Of Immobilized Enzymes In the Cfmsmentioning

confidence: 99%

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Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System

et al. 2019

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Immobilized microfluidic enzyme reactors (IMER) are of particular interest for automation of enzyme cascade reactions. Within an IMER, substrates are converted by paralleled immobilized enzyme modules and intermediate products are transported for further conversion by subsequent enzyme modules. By optimizing substrate conversion in the spatially separated enzyme modules purification of intermediate products is not necessary, thus shortening process time and increasing space-time yields. The IMER enables the development of efficient enzyme cascades by combining compatible enzymatic reactions in different arrangements under optimal conditions and the possibility of a cost-benefit analysis prior to scale-up. These features are of special interest for automation of enzymatic glycan synthesis. We here demonstrate a compartmented flow microreactor system using six magnetic enzyme beads (MEBs) for the synthesis of the non-sulfated human natural killer cell-1 (HNK-1) glycan epitope. MEBs are assembled to build compartmented enzyme modules, consisting of enzyme cascades for the synthesis of uridine 5'-diphospho-α-d-galactose (UDP-Gal) and uridine 5'-diphospho-α-d-glucuronic acid (UDP-GlcA), the donor substrates for the Leloir glycosyltransferases β4-galactosyltransferase and β3-glucuronosyltransferase, respectively. Glycan synthesis was realized in an automated microreactor system by a cascade of individual enzyme module compartments each performing under optimal conditions. The products were analyzed inline by an MS-system connected to the microreactor. The high synthesis yield of 96% for the non-sulfated HNK-1 glycan epitope indicates the excellent performance of the automated enzyme module cascade. Furthermore, combinations of other MEBs for nucleotide sugars synthesis with MEBs of glycosyltransferases have the potential for a fully automated and programmed glycan synthesis in a compartmented flow microreactor system.

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Section: Methodsmentioning

confidence: 99%

Section: Activity Assay Of Immobilized Enzymes In the Cfmsmentioning

confidence: 99%

Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System

et al. 2019

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“…By moving the magnet array to the tube, magnetic beads used in a reaction compartment can be fixed and, by that, separated from surrounding fluid. Further details of the reactor device have been previously described (Hübner et al, 2017).…”

Section: Methodsmentioning

confidence: 99%

“…To characterize the activity of the β4GalT -particles in the microreactor system with regard to pH, temperature, and loading, magnetic enzyme immobilisates (MEI) were pumped into the reaction tubing of the system. The MEI were separated from the storage buffer (as described before; Hübner et al, 2017), resuspended in 100 μL of a preheated reaction buffer compartment and pumped to the temperature control module (TCM). Details of the IR-unit for preheating and the TCM are discussed in section describing the modified reactor system.…”

Section: Methodsmentioning

confidence: 99%

“…Magnetic particles with immobilized enzyme are already used in analytical biotechnology as biochemical detectors for sugar (Nomura et al, 2004) and protein (Choi et al, 2002). In a previous article, we introduced a novel microfluidic reactor device allowing contactless separation and resuspension of magnetic particles in combination with segmented microfluidics (Hübner et al, 2015, 2017). By the use of automated syringe pumps and multiport valves the device creates aqueous compartments in disposable tubings as reaction compartments with volumes around 10 to 250 μL.…”

Section: Introductionmentioning

confidence: 99%

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A Compartmented Flow Microreactor System for Automated Optimization of Bioprocesses Applying Immobilized Enzymes

Heinzler

Hübner

Fischöder

et al. 2018

Front. Bioeng. Biotechnol.

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In the course of their development, industrial biocatalysis processes have to be optimized in small-scale, e. g., within microfluidic bioreactors. Recently, we introduced a novel microfluidic reactor device, which can handle defined reaction compartments of up to 250 μL in combination with magnetic micro carriers. By transferring the magnetic carriers between subsequent compartments of differing compositions, small scale synthesis, and bioanalytical assays can be conducted. In the current work, this device is modified and extended to broaden its application range to the screening and optimization of bioprocesses applying immobilized enzymes. Besides scaling the maximum compartment volume up to 3 mL, a temperature control module, as well as a focused infrared spot were integrated. By adjusting the pump rate, compartment volumes can be accurately dosed with an error <5% and adjusted to the requested temperature within less than a minute. For demonstration of bioprocess parameter optimization within such compartments, the influence of pH, temperature, substrate concentration, and enzyme carrier loading was automatically screened for the case of transferring UDP-Gal onto N-acetylglucosamine linked to a tert-butyloxycarbonyl protected amino group using immobilized β1,4-galactosyltransferase-1. In addition, multiple recycling of the enzyme carriers and the use of increased compartment volumes also allows the simple production of preparative amounts of reaction products.

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Small tools for sweet challenges: advances in microfluidic technologies for glycan synthesis

Pinnock

Daniel

2022

Anal Bioanal Chem

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An automated and compartmented fluidic reactor device for multi-step sample-to-answer processes using magnetic particles

Abstract: A benchtop device that combines segmented flow with magnetic particle separation and active resuspension capabilities for biotechnological applications, e.g. biomolecule purification.

Cited by 4 publications

References 43 publications

Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System

Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System

A Compartmented Flow Microreactor System for Automated Optimization of Bioprocesses Applying Immobilized Enzymes

Small tools for sweet challenges: advances in microfluidic technologies for glycan synthesis

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