Digital Microfluidics-Enabled Analysis of Individual Variation in Liver Cytochrome P450 Activity
| Autor: | Markus Haapala, Gowtham Sathyanarayanan, Tiina Sikanen |
|---|---|
| Přispěvatelé: | Division of Pharmaceutical Chemistry and Technology, Drug Research Program |
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
116 Chemical sciences
Context (language use) 02 engineering and technology Analytical Chemistry 03 medical and health sciences Liver disease Cytochrome P-450 Enzyme System Lab-On-A-Chip Devices medicine Humans Digital microfluidics 030304 developmental biology Enzyme Assays chemistry.chemical_classification 0303 health sciences Chemistry Cytochrome P450 activity Equipment Design 021001 nanoscience & nanotechnology medicine.disease Enzymes Immobilized Thermal control Enzyme Biochemistry Liver Microsome 0210 nano-technology Function (biology) |
| Popis: | The superfamily of hepatic cytochrome P450 (CYP) enzymes is responsible for the intrinsic clearance of the majority of therapeutic drugs in humans. 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 interindividual variation in one of the four model reaction activities, that is, ethoxyresorufin O-deethylation (CYP1A1/2), between five donors. Overall, the developed protocol consumes only about 15 mu 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|