Modular Microfluidic System for Emulation of Human Phase I/Phase II Metabolism

Abstract

We present a microfluidic device for coupled phase I/phase II metabolic reactions in vitro. The chip consists of microchannels, which are used as packed bed reactor compartments, filled with superparamagnetic microparticles bearing recombinant microsomal phase I cytochrome P450 or phase II conjugating enzymes (UDP-glucuronosyltransferase). Online coupling of the microfluidic device with LC/MS enabled the quantitative assessment of coupled phase I/phase II transformations, as demonstrated for two different substrates, 7-benzyloxy-4-trifluoromethylcoumarin (BFC) and dextromethorphan (DEX). In contrast, conventional sequential one-pot incubations did not generate measurable amounts of phase II metabolites. Because the microfluidic device is readily assembled from standard parts and can be equipped with a variety of recombinant enzymes, it provides a modular platform to emulate and investigate hepatic metabolism processes, with particular potential for targeted small-scale synthesis and identification of metabolites formed by sequential action of specific enzymes.

An Immobilized Enzyme Reactor for Spatiotemporal Control over Reaction Products

“…An important feature of multienzyme cascade systems is the ability to precisely control the order of each reaction. To demonstrate the importance of maintaining correct enzyme order in a reaction sequence, one study observed a ≈200‐fold increase in product yield when two enzymes were correctly ordered along a fluidic stream . Of particular interest are methods that use site‐selective immobilization to colocalize multiple enzymes on the same chip for circumventing the need to separate each reaction in different compartments, as demonstrated by Niemeyer and co‐workers using DNA hybridization .…”

Self‐Immobilizing Oxidoreductases for Flow Biocatalysis in Miniaturized Packed‐Bed Reactors

“…The microfluidic packed‐bed reactor consists of a PMMA chip that contains four microchannels, each with a volume of about 10 μL (Fig. ) . The chip had rectangular Nd magnets located underneath the microchannels to retain the superparamagnetic beads, which were loaded by simple infusion of a bead suspension.…”

The Promises and the Challenges of Biotransformations in Microflow

“…Elimination of high‐throughput process parameters strongly depends on the integration of process analytics in microfluidic toolboxes, such as UV/Vis, infrared (IR), near infrared spectroscopy (NIR), mass spectrometry (MS) or Raman spectroscopy, as well as NMR . Monitoring of substrate/product concentration at the outlet from the microreactor using high‐performance liquid chromatography (HPLC) or HPLC‐MS enabled quenching and dilution, or injection of internal standards using the switching valves. Apart from frequently used fluorescence microscopy, thermal lens microscopy allows for ultra precise detection of single molecules in liquids based on optical absorption measurements, also enabling on‐line model validation of molecular transport in a liquid–liquid microflow .…”

“…Increased biocatalyst loads in microreactors could be obtained either by immobilization in surface‐attached thin layers of hydrogels (Figure c), on surface‐integrated nanostructures such as nanosprings (Figure g), or by the use of moving unsinkable graphene sheets, where biocatalyst reuse is provided by centrifugation of the outflow fluid . Further introduction of nanomaterials including biomimetic structures in microreactors, together with the genetic introduction of tags in bacterial cells and enzymes, offers the possibility for more specific immobilization and spatial organization of biocatalysts at specific sites, especially using DNA nanotechnology as a programmable tool for engineering multienzyme catalysis . Microflow format was found beneficial also for packed‐bed reactors (Figures f and e) with biocatalysts immobilized in porous beads, on streptavidin‐coated superparamagnetic microbeads, in electrospun nanomats, or in various hydrogels, recently also prepared as alginate‐silica hybrids, which have the advantages of homogenous structure, better stability, and nontoxicity, and are injectable .…”

“…Further introduction of nanomaterials including biomimetic structures in microreactors, together with the genetic introduction of tags in bacterial cells and enzymes, offers the possibility for more specific immobilization and spatial organization of biocatalysts at specific sites, especially using DNA nanotechnology as a programmable tool for engineering multienzyme catalysis . Microflow format was found beneficial also for packed‐bed reactors (Figures f and e) with biocatalysts immobilized in porous beads, on streptavidin‐coated superparamagnetic microbeads, in electrospun nanomats, or in various hydrogels, recently also prepared as alginate‐silica hybrids, which have the advantages of homogenous structure, better stability, and nontoxicity, and are injectable . Miniaturized packed‐bed reactors enabled not only very high enzyme loads, but also very good accessibility and operational stability of biocatalysts due to diminished fluid flow anomalies such as back mixing and dead zones, and very low‐pressure drop …”