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A combination of a microfluidic device with a light modulation system was developed to detect the oxygen consumption rate (OCR) of a single developing zebrafish embryo via phase-based phosphorescence lifetime detection. The microfluidic device combines two components: an array of glass microwells containing Pt(II) octaethylporphyrin as an oxygen-sensitive luminescent layer and a microfluidic module with pneumatically actuated glass lids above the microwells to controllably seal the microwells of interest. The total basal respiration (OCR, in pmol O2/min/embryo) of a single developing zebrafish embryo inside a sealed microwell has been successfully measured from the blastula stage (3 h post-fertilization, 3 hpf) through the hatching stage (48 hpf). The total basal respiration increased in a linear and reproducible fashion with embryonic age. Sequentially adding pharmacological inhibitors of bioenergetic pathways allows us to perform respiratory measurements of a single zebrafish embryo at key developmental stages and thus monitor changes in mitochondrial function in vivo that are coordinated with embryonic development. We have successfully measured the metabolic profiles of a single developing zebrafish embryo from 3 hpf to 48 hpf inside a microfluidic device. The total basal respiration is partitioned into the non-mitochondrial respiration, mitochondrial respiration, respiration due to adenosine triphosphate (ATP) turnover, and respiration due to proton leak. The changes in these respirations are correlated with zebrafish embryonic development stages. Our proposed platform provides the potential for studying bioenergetic metabolism in a developing organism and for a wide range of biomedical applications that relate mitochondrial physiology and disease.

Metabolic profile analysis of a single developing zebrafish embryo via monitoring of oxygen consumption rates within a microfluidic device