Measurement of Dissolved Oxygen in Highly Restricted (PIcoliter) Volumes Utilizing Thin Film Luminescent Sensors

Abstract

Researchers at the Microscale Life Sciences Center undertook an ambitious project to examine the metabolic and genomic processes in single mammalian cells related to esophageal adenocarcinomas in Barrett's Esophagus patients. Examination of one cell at a time was necessitated by the need to resolve questions related to cellular heterogeneity and thus required that experiments be conducted in highly restricted (picoliter) volumes. Measurement of dissolved oxygen is a key parameter in understanding cellular metabolism. This need to examine oxygen consumption rates of single mammalian cells motivated the development of new sensors and new methods for the measurement of dissolved oxygen in picoliter volumes. Commercially available luminescent sensors as well as in-house customized luminescent sensors were characterized. Key parameters that were characterized included sensor deposition into microwells, sensor lifetime measurement for dry and wet sensors, sensor adhesion assessment, photodegradation studies, and measurement of singlet oxygen emission signals. This characterization work led to the development of a new lifetime measurement method for luminescent sensors, dubbed Long-Pulsed Luminescence (LPL). Application of this new method determined that a modified platinum porphyrin sensor was the best performer in terms of analytic usefulness, but the best application-driven sensor was a commercially available microsphere product, which also utilized a platinum porphyrin reporter chemistry.