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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 for imaging enzyme activities. a Chemical structures of 1,2-dioxetane chemiluminescent substrates and their activation by hydrolytic enzymes. Galacton-plus is a substrate for β-galatosidase, and CSPD is an alkaline phosphatase substrate. The hydrolytic activity of β-galatosidase or alkaline phosphatase removes the protective group X and results in substrate activation. b In the absence of a recipient fluorescent dye, the activated substrate self-decomposes and results in blue chemiluminescence emission or heat release due to water quenching. c In the presence of a fluorescent recipient, the activated substrate can directly transfer its chemical energy via formation of a charge-transfer complex with the recipient fluorophore
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Fig1: CIEEL energy transfer for imaging enzyme activities. a Chemical structures of 1,2-dioxetane chemiluminescent substrates and their activation by hydrolytic enzymes. Galacton-plus is a substrate for β-galatosidase, and CSPD is an alkaline phosphatase substrate. The hydrolytic activity of β-galatosidase or alkaline phosphatase removes the protective group X and results in substrate activation. b In the absence of a recipient fluorescent dye, the activated substrate self-decomposes and results in blue chemiluminescence emission or heat release due to water quenching. c In the presence of a fluorescent recipient, the activated substrate can directly transfer its chemical energy via formation of a charge-transfer complex with the recipient fluorophore

Mentions: In addition to peroxalate-based compounds, several 1,2-dioxetane chemiluminescent substrates have been widely used for biochemical detection of enzyme activities in vitro such as β-galactosidase (Galacton-plus®, Fig. 1a) and alkaline phosphatase (CSPD®, Fig. 1a) [16]. Upon removal of the protective galactosyl or phosphoryl group, the negative electrical charge on the phenolate group triggers an electron transfer event that in turn induces decomposition of the high-energy 1,2-dioxetane ring (Fig. 1a) [16]. The decomposition would generate several high-energy intermediates in excited states. One possibility is generating an excited methyl-benzene carboxylate moiety. The fates of the excited intermediates would largely depend upon the surrounding environments. In an aqueous solution, the majority of excited intermediates are quenched by surrounding water molecules and results in heat release (Fig. 1b) [16]. It is unavoidable to have heat release during CIEEL imaging due to the ubiquitous presence of water in biological systems. Nevertheless, the extent of water quenching should not vary considerably among different animal subjects as water activity tends to be well controlled in tissues. Reducing water activity by adding macromolecule enhancers, such as polymer micelles, protects them from water quenching and thus promotes energy released through the phenolate ring as blue chemiluminescence (466 nm, Fig. 1b). However, blue light is not suitable for in vivo imaging due to its strong absorbance by hemoglobin. Redder emission can be achieved by coupling the activated substrate with a nearby recipient fluorophore. The activated substrate can directly transfer its chemical energy to the fluorescent dye via intermolecular CIEEL energy transfer (Fig. 1c). Of note, it is possible to use a near-infrared fluorescent probe as the energy recipient in order to further enhance tissue penetration and signal strength. In this study, we examine the use of 1,2-dioxetane substrates for visualizing enzyme activities in biological systems. In particular, we demonstrate the use of this novel concept for imaging increased alkaline phosphatase activity associated with tissue inflammation.Fig. 1


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

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

CIEEL energy transfer for imaging enzyme activities. a Chemical structures of 1,2-dioxetane chemiluminescent substrates and their activation by hydrolytic enzymes. Galacton-plus is a substrate for β-galatosidase, and CSPD is an alkaline phosphatase substrate. The hydrolytic activity of β-galatosidase or alkaline phosphatase removes the protective group X and results in substrate activation. b In the absence of a recipient fluorescent dye, the activated substrate self-decomposes and results in blue chemiluminescence emission or heat release due to water quenching. c In the presence of a fluorescent recipient, the activated substrate can directly transfer its chemical energy via formation of a charge-transfer complex with the recipient fluorophore
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: CIEEL energy transfer for imaging enzyme activities. a Chemical structures of 1,2-dioxetane chemiluminescent substrates and their activation by hydrolytic enzymes. Galacton-plus is a substrate for β-galatosidase, and CSPD is an alkaline phosphatase substrate. The hydrolytic activity of β-galatosidase or alkaline phosphatase removes the protective group X and results in substrate activation. b In the absence of a recipient fluorescent dye, the activated substrate self-decomposes and results in blue chemiluminescence emission or heat release due to water quenching. c In the presence of a fluorescent recipient, the activated substrate can directly transfer its chemical energy via formation of a charge-transfer complex with the recipient fluorophore
Mentions: In addition to peroxalate-based compounds, several 1,2-dioxetane chemiluminescent substrates have been widely used for biochemical detection of enzyme activities in vitro such as β-galactosidase (Galacton-plus®, Fig. 1a) and alkaline phosphatase (CSPD®, Fig. 1a) [16]. Upon removal of the protective galactosyl or phosphoryl group, the negative electrical charge on the phenolate group triggers an electron transfer event that in turn induces decomposition of the high-energy 1,2-dioxetane ring (Fig. 1a) [16]. The decomposition would generate several high-energy intermediates in excited states. One possibility is generating an excited methyl-benzene carboxylate moiety. The fates of the excited intermediates would largely depend upon the surrounding environments. In an aqueous solution, the majority of excited intermediates are quenched by surrounding water molecules and results in heat release (Fig. 1b) [16]. It is unavoidable to have heat release during CIEEL imaging due to the ubiquitous presence of water in biological systems. Nevertheless, the extent of water quenching should not vary considerably among different animal subjects as water activity tends to be well controlled in tissues. Reducing water activity by adding macromolecule enhancers, such as polymer micelles, protects them from water quenching and thus promotes energy released through the phenolate ring as blue chemiluminescence (466 nm, Fig. 1b). However, blue light is not suitable for in vivo imaging due to its strong absorbance by hemoglobin. Redder emission can be achieved by coupling the activated substrate with a nearby recipient fluorophore. The activated substrate can directly transfer its chemical energy to the fluorescent dye via intermolecular CIEEL energy transfer (Fig. 1c). Of note, it is possible to use a near-infrared fluorescent probe as the energy recipient in order to further enhance tissue penetration and signal strength. In this study, we examine the use of 1,2-dioxetane substrates for visualizing enzyme activities in biological systems. In particular, we demonstrate the use of this novel concept for imaging increased alkaline phosphatase activity associated with tissue inflammation.Fig. 1

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