Activatable Raman Probes Utilizing Enzyme-Induced Aggregate Formation for Selective Ex Vivo Imaging

Abstract

Detecting multiple enzyme activities simultaneously with high spatial specificity is a promising strategy to investigate complex biological phenomena, and Raman imaging would be an excellent tool for this purpose due to its high multiplexing capabilities. We previously developed activatable Raman probes based on 9CN-pyronins, but specific visualization of cells with target enzyme activities proved difficult due to leakage of the hydrolysis products from the target cells after activation. Here, focusing on rhodol bearing a nitrile group at the position of 9 (9CN-rhodol), we established a novel mechanism for Raman signal activation based on a combination of aggregate formation (to increase local dye concentration) and the resonant Raman effect along with the bathochromic shift of the absorption, and utilized it to develop Raman probes. We selected the 9CN-rhodol derivative 9CN-JCR as offering a suitable combination of increased stimulated Raman scattering (SRS) signal intensity and high aggregate-forming ability, resulting in good retention in target cells after probe activation. By using isotope-edited 9CN-JCR-based probes, we could simultaneously detect β-galactosidase, γ-glutamyl transpeptidase, and dipeptidyl peptidase-4 activities in live cultured cells and distinguish cell regions expressing target enzyme activity in Drosophila wing disc and fat body ex vivo.

Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

“…We also obtained a Raman image of intracellular and extracellular water distributions, using the Raman signal that indicates ice crystal in and out of cryopreserved cells, which can be useful for understandings in the conditions of cryopreserved cells [41]. These results demonstrate the potential of cryo-Raman microscopy to detect the chemical response of endogenous molecules from their Raman spectra while using various Raman tags and probes [14],[17],[42] to perform highly multiplex observations.…”

Activity-Based Fluorescence Diagnostics for Cancer

“…In addition to those highlighted here, other fluorescence probes targeting different biomarkers or fluorescence probes based on different detection mechanisms such as aggregation-induced emission (AIE) , and excited-state intramolecular proton transfer (ESIPT) , have been also developed. Moreover, activatable probes based on other technologies such as chemiluminescence, − photoacoustic imaging ,− and Raman imaging , have been reported in recent years. More sophisticated contrast probes utilizing these technologies can be anticipated.…”

Triple-Bond Vibrations: Emerging Applications in Energy and Biological Sciences

“…In a different application, they combined probe concentration increase via aggregate formation, the resonance enhancement effect, and the absorption spectrum shift to activate the nitrile Raman signal upon substrate cleavage. 103 Specifically, they used a nitrile-containing rhodol (at position 9) as the Raman tag and the isotopic editing strategy to yield four different probing frequencies. They demonstrated their method by simultaneously detecting β-galactosidase, γglutamyl transpeptidase, and dipeptidyl peptidase-4 activities in live cultured cells.…”

“…Specifically, they used the strategy of electronic preresonance (EPR) to enhance the Raman signal, which was achieved upon reaction between the substrate and the targeted enzyme, and the strategy of isotopic editing to obtain four different nitrile stretching frequencies (Figure ). In a different application, they combined probe concentration increase via aggregate formation, the resonance enhancement effect, and the absorption spectrum shift to activate the nitrile Raman signal upon substrate cleavage . Specifically, they used a nitrile-containing rhodol (at position 9) as the Raman tag and the isotopic editing strategy to yield four different probing frequencies.…”