A practical and affordable photometric method for routine low volume verification of fixed-head automated liquid...

Accurate microliter-level dispensing is crucial for generating reliable and reproducible results in biomedical and diagnostic assays. While automated liquid handling systems (ALHS) improve throughput and reduce operator variability, volumetric accuracy (trueness and precision) can drift with liquid properties, tip geometry, and instrument settings, necessitating routine verification. This study utilized ALHS from Agilent Technologies, Bravo automated liquid handling platform (Bravo) with a 96-barrel head and evaluated its dispensing performance against a calibrated manual pipetting reference. We tested four settings of the Bravo, combining two tip capacities (30 µL and 70 µL) with two aspiration modes (with or without post-aspirate air gap volume, PAV) to determine optimal accuracy. Volumetric accuracy was checked every 10 days over 30 days, comparing performance before and after preventive maintenance of the 96-barrel head. Bravo pipetting demonstrated excellent agreement with manual pipetting (r² = 0.998; p < 0.0001), validating the reliability of the spectrophotometric approach. Although the volumetric accuracy was generally stable with Bravo, a slight tendency toward under-dispensing was noticed compared to manual pipetting. Among all the tested conditions, the 30 µL tip with PAV achieved the highest trueness (-2.22% bias) and precision (CV ≤ 1%) for 4 µL target volumes. Furthermore, longitudinal assessments revealed no significant change in mean dispensed volumes before or after preventive maintenance. In this study, the overall performance of the Bravo remained within acceptable operational limits. We believe the Orange-G protocol described here provides a fit-for-purpose alternative to commercial verification systems, enabling regular, plate-level checks aligned with concepts of volumetric performance in automated systems.