Validated workflows for preparing and characterizing core-stained and surface-labeled fluorescent polymer particles...

Preparation workflows of fluorescent nm- and µm-sized polymer particles used as reporters in fluorescent assays, bioimaging, and sensing studies or calibration tools for fluorescence methods in the life sciences can be time-consuming and labor-intensive. Also, the outcome can be operator dependent. Here, we present simple and cost-efficient automated workflows for dye loading and surface labeling of polystyrene particles (PSP), using a commercial self-programmable pipetting robot. For developing and fine-tuning automated staining workflows, hydrophobic Nile Red (NR) was incorporated into 100 nm, 200 nm, and 1000 nm PSP by an adapted swelling procedure of premanufactured particles in the presence of hydrophobic dyes, thereby confirming its reliability and versatility. Subsequent expansion of our automation concept to the labeling of carboxylated PSP with pH-sensitive 6-aminofluorescein (6-AMF) and aminated PSP with pH-responsive fluorescein isocyanate (FITC) demonstrated its broad applicability. All automated workflows were optimized and validated by gravimetry and spectroscopic measurements with a microtiter plate (MTP) reader in absorption and fluorescence mode to ensure particle recovery and reproducible fluorescence features and determine PSP dye loading and labeling efficiencies. Comparison with the manual fabrication of NR-stained PSP using an established swelling protocol showed that our automation approach utilizing a pipetting robot considerably reduced the variability in particle recovery and dye loading efficiency. Overall, our simple, labor- and time-efficient workflows with inexpensive and broadly available commercial automation tools present attractive alternatives to manual particle loading and labeling and provide the basis for fast parameter screening, parallel processing, and decreased hands-on time.