The quantitative determination of proteins in picoliter-volume vials is described. The assay is based on the bioluminescence of the photoprotein aequorin along with photon-counting detection. Using this approach, avidin can be detected at femtomole levels by taking advantage of its inhibitory effect on the bioluminescence signal generated by biotinylated recombinant aequorin. The picoliter vials were fabricated on glass substrates using a laser ablation technique. Parameters that affect the reproducibility of the assay such as the fabrication and calibration of the pipets, the fabrication of the vials, and the composition of the assay solutions were studied.

A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.

A new method for calibrating micropipets and determining accurate injection volumes using a pressure-based injector has been developed. This method employs the bioluminescent protein aequorin and can be used to determine injection volumes as small as 3 pL. The calibration plots are linear over at least 3 orders of magnitude. In contrast to conventional micropipet calibration methods that employ fluorescent molecules, the present method produces small background signals.

The ability to detect biomolecules in single cells is important in order to fully understand the processes by which many biochemical events occur. To that end, we have developed a bioluminescence binding assay capable of measuring the intracellular biotin content of individual cells. The assay depends on competition between an aequorin-biotin conjugate (AEQ-biotin) and free biotin within the oocytes for binding sites on the protein avidin. The assay is performed by microinjecting each component into the oocytes and following the resulting bioluminescence within the oocyte upon triggering of aequorin. Results obtained using sea urchin oocytes show that the assay performed within the cells behaves in a manner consistent with assay theory. Using the assay, the individual biotin content of the oocytes is an average of approximately 20 amol. To our knowledge, this is the first reported multicomponent binding assay to be performed inside an intact single cell.