Human-Serum Validation of Engineered Enzymes for PET Microplastic Degradation
Serum time-course (37 °C): Incubate PET microplastics with engineered PET-degrading enzymes (PETase, MHETase) alongside controls (enzyme-only, plastic-only, serum blanks). Pre-define sampling times.Quench & prep: Stop reactions and prepare extracts for analysis using standard lab procedures.Chemistry readout (primary): Quantify PET breakdown products (TPA, MHET, BHET, EG) by HPLC/MS with external calibration, internal standards, and routine QC.Particle counts (supportive): Pre/post microplastic particle enumeration (e.g., flow-imaging or filter-image methods) with size-bin summaries and bead/reference checks.Polymer confirmation (confirmatory): O-PTIR (± FTIR/Raman) to corroborate PET identity and surface-chemistry changes.Enzyme stability: Measure residual activity over time in serum.Cell safety: HEK293 viability/cytotoxicity dose-response; include byproduct controls (terephthalic acid, ethylene glycol).Reproducibility & analysis: Replicates across serum lots/days; pre-set acceptance criteria and a registered analysis plan (mixed-effects models). Go/no-go decisions tied to activity, stability, and safety outcomes.