Fabrication of dye‐loaded microparticles as substrates for PETase assay

Abstract Polyethylene terephthalate (PET) poses significant challenges to waste management. The discovery of PET‐degrading enzymes, such as PETase, has prompted efforts in the engineering of new PETase variants with improved activities. However, the assessment of the variant activities relies on either proxy substrates or di‐/tri‐units of the monomers which result in false positives. Furthermore, the detection of enzymatic depolymerization products of PET into their monomers terephthalic acid and ethylene glycol has relied on HPLC which requires extensive preparation steps. This work introduces a realistic substrate for a fluorescent detection method by utilizing rhodamine‐B‐loaded PET microparticles ((RhB)mPETs) to evaluate PETase activity. (RhB)mPETs are fabricated by the emulsion solvent evaporation method. Upon degradation of the mPET, RhB is released and can be detected fluorimetrically. Optimization of the experimental protocols identifies sodium dodecyl sulfate as an efficient agent to increase signal by reducing RhB rebinding to the polymer upon release, albeit with a trade‐off in PET degradation rate. This is due to an increase in polymer crystallinity in the fabricated (RhB)mPETs. The increased crystallinity impacts the enzymatic depolymerization rate and reduces the sensitivity of the assay. Challenges posed by (RhB)mPETs need to be addressed in order for the use of developed mPETs as substrates in evaluating engineered PETase. © 2026 Society of Chemical Industry.