An immunoassay using aequorin as a bioluminescent label to measure femtomole levels of intracellular biomolecules

Characterizing the activities of cells requires the identification and quantification of intracellular chemical changes of several substances. Conventional biochemical analyses measure an average composition of thousands, possibly millions, of cells, yet the distribution of substances among these cells varies within a given population. Therefore, there is a need for bioanalytical procedures that are capable of measuring the biochemical composition of single cells. It has been demonstrated that the Ca²⁺-triggered luminescence reaction of biotinylated recombinant aequorin can be inhibited by the presence of avidin. This inhibition in bioluminescence intensity can be used to determine the biotin concentration in a sample. This assay was initially developed to detect concentrations as low as 10^-14 M in a sample size of 100 ML. This technology is scaled down to be used inside a single mammalian fibroblast cell with a volume of approximately 4 pL. It has been demonstrated that recombinant aequorin is well suited for use as a label in highly sensitive assays, which can be performed in a single cell.