In vivo imaging of endogenous enzyme activities using luminescent 1,2-dioxetane compounds

Abstract

BackgroundHere we present a non-invasive imaging method for visualizing endogenous enzyme activities in living animals. This optical imaging method is based on an energy transfer principle termed chemically initiated electron exchange luminescence (CIEEL). The light energy is provided by enzymatic activation of metastable 1,2-dioxetane substrates, whose protective groups are removed by hydrolytic enzymes such as β-galactosidase and alkaline phosphatase. In the presence of a nearby fluorescent recipient, the chemical energy within the activated substrate is then transferred via formation of a charge-transfer complex with the fluorophore, a mechanism closely related to glow stick chemistry.ResultsEfficient CIEEL energy transfer requires close proximity between the trigger enzyme and the fluorescent recipient. Using cells stained with fluorescent dialkylcarbocyanines as the energy recipients, we demonstrated CIEEL imaging of cellular β-galactosidase or alkaline phosphatase activity. In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity.ConclusionsIn this report, we provide proof-of-concept for CIEEL imaging of in vivo enzymatic activity. In addition, we demonstrate the use of CIEEL energy transfer for visualizing elevated alkaline phosphatase activity associated with tissue inflammation in living animals.

In Vivo Delivery and Activation of Masked Fluorogenic Hydrolase Substrates by Endogenous Hydrolases in C. elegans

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Activatable Chemiluminescent Molecular Probes for Bioimaging and Biosensing

“…made a significant breakthrough and extended the applications of Schaap's dioxetane‐based chemiluminescence to bioimaging and biosensing in vivo [82] . However, the extremely low chemiluminescence signal was observed under physiological conditions due to the quenching effect of water molecules [83] . To amplify the chemiluminescence emission signal, the researchers have adopted the following methods: (1) the formation of polymers, in which the dioxetane unit used as a polymer monomer [84] .…”

Overview of Optical and Electrochemical Alkaline Phosphatase (ALP) Biosensors: Recent Approaches in Cells Culture Techniques

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“…Specifically, live-cell imaging with optical microscopy methods is powerful because they enable real-time detection of cellular processes [60]. Optical events are detected using bioluminescent and fluorescent probes that emit radiation at near-infrared or visible wavelengths, which are detected using optical cameras [60]. Optical methods have been used in detecting analytes because of their simple application.…”