We report the development and characterization of digital microfluidic (DMF) immobilized enzyme reactors (IMERs) for studying cytochrome P450 (CYP)-mediated drug metabolism on droplet scale. The on-chip IMERs consist of porous polymer (thiol-ene) monolith plugs prepared in situ by photopolymerization and functionalized with recombinant CYP1A1 isoforms (an important detoxification route for many drugs and other xenobiotics). The DMF devices also incorporate inexpensive, inkjet-printed microheaters for on-demand regio-specific heating of the IMERs to physiological temperature, which is crucial for maintaining the activity of the temperature-sensitive CYP reaction. For on-chip monitoring of the CYP activity, the DMF devices were combined with a commercial well-plate reader, and a custom fluorescence quantification method was developed for detection of the chosen CYP1A1 model activity (ethoxyresorufin-O-deethylation). The reproducibility of the developed assay was examined with the help of ten parallel CYP-IMERs. All CYP-IMERs provided statistically significant difference (in fluorescence response) compared to any of the negative controls (including room-temperature reactions). The average (n = 10) turnover rate was 20.3 ± 9.0 fmol resorufin per minute. Via parallelization, the concept of the droplet-based CYP-IMER developed in this study provides a viable approach to rapid and low-cost prediction of the metabolic clearance of new chemical entities in vitro.Electronic supplementary materialThe online version of this article (10.1007/s00216-018-1280-7) contains supplementary material, which is available to authorized users.

It is shown here that controlled mixing of a gelator, drug, solvent, and antisolvent in a microfluidic channel leads to faster setting gels and more robust materials with longer release profiles than the physical gels of the same composition obtained using random mixing in solution. The system is similar to a related gelator system we had studied previously, but we were unable to apply the same gelling procedure because of the instability of the colloid caused by the small structural modification (length of the alkyl chain in the bis-imidazolium head group). This situation holds true for the gels formed with varying compositions and under different conditions (gelator/drug ratio, solvent proportion, and flow rates), with the most significant differences being the improved gel rheology and slower drug release rates. Very importantly, the gels (based on a previously unexplored system) have a higher water content ratio (water/EtOH 4:1) than others in the family, making their medicinal application more attractive. The gels were characterized by a variety of microscopy techniques, X-ray diffraction and infrared spectroscopy, and rheology. Salts of the antiinflammatory drugs ibuprofen and indomethacin were successfully incorporated into the gels. The diffraction experiments indicate that these composite gels with relatively short alkyl chains in the gelator component contrast to previous systems, in that they exhibit structural order and the presence of crystalline areas of the drug molecule implying partial phase separation (even though these drug crystallites are not discernible by microscopy). Furthermore, the release study with the gel incorporating ibuprofenate showed promising results that indicate a possible drug delivery vehicle application for this and related systems.

This work describes the interfacing of electrowetting-on-dielectric based digital microfluidic (DMF) sample preparation devices with ambient mass spectrometry (MS) via desorption atmospheric pressure photoionization (DAPPI). The DMF droplet manipulation technique was adopted to facilitate drug distribution and metabolism assays in droplet scale, while ambient mass spectrometry (MS) was exploited for the analysis of dried samples directly on the surface of the DMF device. Although ambient MS is well-established for bio- and forensic analyses directly on surfaces, its interfacing with DMF is scarce and requires careful optimization of the surface-sensitive processes, such as sample precipitation and the subsequent desorption/ionization. These technical challenges were addressed and resolved in this study by making use of the high mechanical, thermal, and chemical stability of SU-8. In our assay design, SU-8 served as the dielectric layer for DMF as well as the substrate material for DAPPI-MS. The feasibility of SU-8 based DMF devices for DAPPI-MS was demonstrated in the analysis of selected pharmaceuticals following on-chip liquid-liquid extraction or an enzymatic dealkylation reaction. The lower limits of detection were in the range of 1–10 pmol per droplet (0.25–1.0 µg/mL) for all pharmaceuticals tested.

mixed, and split in parallel to perform sequential sample manipulation protocols, such as rinsing, preconcentration, reaction, and extraction. Interfacing of DMF with a range of optical, [3,4] electrochemical, [5-7] and mass spectrometric detection [8-11] methods is well established and the fabrication of the devices has already been pushed toward low-cost cleanroom-free techniques. [12,13] As a result, DMF is a robust and cost-effective technology for miniaturization of various biochemical assays including but not limited to immunological, [14] enzymatic, [15] cell-based, [16] PCR, [17] drug, [18] and biopsy [19] screening assays. However, in some instances, the specificity of direct in situ detection may not be sufficient to distinguish multiple sample components from each other, and in these cases, the droplet must be transferred for further analysis into on-chip or off-chip chemical separation systems. To date, much of the prior work has exploited off-chip analysis of DMF-manipulated samples, which is however relatively complicated and prone to sample losses during the droplet transfer from micro-to macro scale. Although a variety of miniaturized electrophoretic and chromatographic separation chips have been reported in general, [20-22] their integration with DMF has not been comprehensively established, likely because of the marked differences in the applicable microfabrication materials and methods. DMF driving electrodes are typically defined on a glass substrate by photolithography. Recently, increasing effort has been put into

The superfamily of hepatic cytochrome P450 (CYP) enzymes is responsible for the intrinsic clearance of the majority of therapeutic drugs in human. However, the kinetics of drug clearance via CYPs varies significantly among individuals due to both genetic and external factors, and the enzyme amount and function are also largely impacted by many liver diseases. In this study, we developed a new methodology, based on digital microfluidics (DMF), for rapid determination of individual alterations in CYP activity from human-derived liver samples in biopsy-scale. The assay employs human liver microsomes (HLMs), immobilized on magnetic beads to facilitate determination of the activity of microsomal CYP enzymes in a parallelized system at physiological temperature. The thermal control is achieved with the help of a custom-designed, inkjet-printed microheater array modularly integrated with the DMF platform. The CYP activities are determined with the help of prefluorescent, enzyme-selective model compounds by quantifying the respective fluorescent metabolites based on optical readout in situ. The selectivity and sensitivity of the assay was established for four different CYP model reactions, and the diagnostic concept was validated by determining the inter-individual variation in one of the four model reaction activities, i.e., ethoxyresorufin O-deethylation (CYP1A1/2), between five donors. Overall, the developed protocol consumes only about 15 g microsomal protein per assay. It is thus technically adaptable to screening of individual differences in CYP enzyme function from biopsy-scale liver samples in an automated fashion, so as to support tailoring of medical therapies, for example, in the context of liver disease diagnosis.