Genetically Modified Semisynthetic Bioluminescent Photoprotein Variants: Simultaneous Dual-Analyte Assay in a Single Well Employing Time Resolution of Decay Kinetics

Abstract

Progress in the miniaturization and automation of complex analytical processes depends largely on increasing the sensitivity, diversity, and robustness of current labels. Because of their ubiquity and ease of use, fluorescent, enzymatic, and bioluminescent labels are often employed in such miniaturized and multiplexed formats, with each type of label having its own unique advantages and drawbacks. The ultrasensitive detection limits of bioluminescent reporters are especially advantageous when dealing with very small sample volumes and biological fluids. However, bioluminescent reporters currently do not have the multiplexing capability that fluorescent labels do. In an effort to address this limitation, we have developed a method of discriminating two semisynthetic aequorin variants from one another using time resolution. In this work we paired two aequorin conjugates with different coelenterazine analogues and then resolved the two signals from one another using the difference in decay kinetics and half-life times. Utilizing this time-resolution, we then developed a simultaneous, dual-analyte, single well assay for 6-keto-prostaglandin-FI-alpha and angiotensin II, two important cardiovascular molecules.

Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in In Vivo Imaging

“…The variants with non-natural amino acid substitutions at both positions, Tyr82 and Trp86, show a mixed influence of both substitutions, with longer emission half-lives as well as greater red-shifted emission spectra. These emission characteristics may allow for multiplex detection by spatial and temporal resolution through the differentiation of the emission wavelength and time [ 51 ].…”

Improving the luminescence properties of aequorin by conjugating to CdSe/ZnS quantum dot nanoparticles: Red shift and slowing decay rate

“…Two last cases can be utilized in dualanalyte assay. Laura Rowe et al using this property distinguished two important cardiovascular factors [69]. More studies are needed to find ways for increasing energy transfer from aequorin to the QD.…”

Fluorescent Protein–photoprotein Fusions and Their Applications in Calcium Imaging

“…Such combinations highlight the flexibility of aequorin when it comes to spectral tuning for multiplexing applications (for more combinations, consult ). In addition to spectral separation, time‐resolved bioluminescence has also been achieved between semisynthetic aequorins with different coelenterazine analogues that showed different decay kinetics . Discrimination of native and i ‐aequorin was done using two separate time intervals, from 0–6 s and from 6–25 s, to resolve two different cardiovascular analyte responses in vitro .…”