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

Tseng JC, Kung AL - J. Biomed. Sci. (2015)

Bottom Line: In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity.In 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.

View Article: PubMed Central - PubMed

Affiliation: Lurie Family Imaging Center, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA, 02215, USA. jct223@gmail.com.

ABSTRACT

Background: Here 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.

Results: Efficient 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.

Conclusions: In 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.

No MeSH data available.


Related in: MedlinePlus

CIEEL energy transfer requires close proximity of the energy source and the recipient fluorophore. a The absorption/emission spectra of Alexa Fluor 647 (red), and the chemiluminescence emission range of Galacton-plus (blue shaded region). b Each well contained equal amounts of biotinylated β-galactosidase for Galacton-plus activation. Increasing levels of streptavidin- or ovalbumin-conjugated Alexa Fluor 647 (0–160 pmol) were added into the wells. The total luminescence output of each well was first visualized without an emission filter (open). The plate was then imaged at 500 nm (self-decomposition) or 680 nm (CIEEL energy transfer). The levels of Alexa Fluor 647 were determined by a fluorescence imaging using an Ex 640/Em 680 nm filter set. c Quantitative representation of the CIEEL energy transfer at 680 nm. Error bar = SEM
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Fig2: CIEEL energy transfer requires close proximity of the energy source and the recipient fluorophore. a The absorption/emission spectra of Alexa Fluor 647 (red), and the chemiluminescence emission range of Galacton-plus (blue shaded region). b Each well contained equal amounts of biotinylated β-galactosidase for Galacton-plus activation. Increasing levels of streptavidin- or ovalbumin-conjugated Alexa Fluor 647 (0–160 pmol) were added into the wells. The total luminescence output of each well was first visualized without an emission filter (open). The plate was then imaged at 500 nm (self-decomposition) or 680 nm (CIEEL energy transfer). The levels of Alexa Fluor 647 were determined by a fluorescence imaging using an Ex 640/Em 680 nm filter set. c Quantitative representation of the CIEEL energy transfer at 680 nm. Error bar = SEM

Mentions: Galacton-plus is a 1,2-dioxetane substrate that has been commonly used in a variety of chemiluminescent assays for detecting β-galactosidase activity in aqueous samples. In order to demonstrate intermolecular CIEEL energy transfer in vitro, we took advantage of biotin-streptavidin interaction to bring together both the bacterial β-galactosidase enzyme and the Alexa Fluor 647 fluorophore (Fig. 2). The biotinylated β-galactosidase served as the trigger enzyme and the streptavidin-conjugated Alexa Fluor 647 functioned as the energy recipient. We reasoned that the biotin-conjugated β-galactosidase would catalyze the 1,2-dioxetane Galacton-plus substrate to release high-energy intermediates. As the biotin-streptavidin interaction brought both moieties in close proximity, the streptavidin-conjugated Alexa Fluor 647 could capture the chemical energy within the intermediates and emit a red luminescence. It is known that resonance energy transfer requires sufficient spectral overlap between the donor’s emission and the recipient's excitation spectra. However, since the Galacton-plus is a blue chemiluminescence substrate, there is virtually no spectral overlap between Galacton-plus’ blue emission and Alexa Fluor’s red excitation spectra (Fig. 2a).Fig. 2


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

Tseng JC, Kung AL - J. Biomed. Sci. (2015)

CIEEL energy transfer requires close proximity of the energy source and the recipient fluorophore. a The absorption/emission spectra of Alexa Fluor 647 (red), and the chemiluminescence emission range of Galacton-plus (blue shaded region). b Each well contained equal amounts of biotinylated β-galactosidase for Galacton-plus activation. Increasing levels of streptavidin- or ovalbumin-conjugated Alexa Fluor 647 (0–160 pmol) were added into the wells. The total luminescence output of each well was first visualized without an emission filter (open). The plate was then imaged at 500 nm (self-decomposition) or 680 nm (CIEEL energy transfer). The levels of Alexa Fluor 647 were determined by a fluorescence imaging using an Ex 640/Em 680 nm filter set. c Quantitative representation of the CIEEL energy transfer at 680 nm. Error bar = SEM
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4477311&req=5

Fig2: CIEEL energy transfer requires close proximity of the energy source and the recipient fluorophore. a The absorption/emission spectra of Alexa Fluor 647 (red), and the chemiluminescence emission range of Galacton-plus (blue shaded region). b Each well contained equal amounts of biotinylated β-galactosidase for Galacton-plus activation. Increasing levels of streptavidin- or ovalbumin-conjugated Alexa Fluor 647 (0–160 pmol) were added into the wells. The total luminescence output of each well was first visualized without an emission filter (open). The plate was then imaged at 500 nm (self-decomposition) or 680 nm (CIEEL energy transfer). The levels of Alexa Fluor 647 were determined by a fluorescence imaging using an Ex 640/Em 680 nm filter set. c Quantitative representation of the CIEEL energy transfer at 680 nm. Error bar = SEM
Mentions: Galacton-plus is a 1,2-dioxetane substrate that has been commonly used in a variety of chemiluminescent assays for detecting β-galactosidase activity in aqueous samples. In order to demonstrate intermolecular CIEEL energy transfer in vitro, we took advantage of biotin-streptavidin interaction to bring together both the bacterial β-galactosidase enzyme and the Alexa Fluor 647 fluorophore (Fig. 2). The biotinylated β-galactosidase served as the trigger enzyme and the streptavidin-conjugated Alexa Fluor 647 functioned as the energy recipient. We reasoned that the biotin-conjugated β-galactosidase would catalyze the 1,2-dioxetane Galacton-plus substrate to release high-energy intermediates. As the biotin-streptavidin interaction brought both moieties in close proximity, the streptavidin-conjugated Alexa Fluor 647 could capture the chemical energy within the intermediates and emit a red luminescence. It is known that resonance energy transfer requires sufficient spectral overlap between the donor’s emission and the recipient's excitation spectra. However, since the Galacton-plus is a blue chemiluminescence substrate, there is virtually no spectral overlap between Galacton-plus’ blue emission and Alexa Fluor’s red excitation spectra (Fig. 2a).Fig. 2

Bottom Line: In living animals, we used a similar approach to non-invasively image alkaline phosphatase activity in the peritoneal cavity.In 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.

View Article: PubMed Central - PubMed

Affiliation: Lurie Family Imaging Center, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA, 02215, USA. jct223@gmail.com.

ABSTRACT

Background: Here 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.

Results: Efficient 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.

Conclusions: In 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.

No MeSH data available.


Related in: MedlinePlus