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Development of a novel lysosome-targetable time-gated luminescence probe for ratiometric and luminescence lifetime detection of nitric oxide in vivo † † Electronic supplementary information (ESI) available: Experimental details for the syntheses of TRP-Tb 3+ and TRP-NO , and supplementary figures. See DOI: 10.1039/c6sc03667h Click here for additional data file.

View Article: PubMed Central - PubMed

ABSTRACT

Trp-notrp-notrp-notrp-notrp-notrp-notrp-no: Rapid, multiplexed, sensitive and specific identification and quantitative detection of nitric oxide (NO) are in great demand in biomedical science. Herein, a novel multifunctional probe based on the intramolecular LRET (luminescence resonance energy transfer) strategy, , was designed for the highly sensitive and selective ratiometric and luminescence lifetime detection of lysosomal NO. Before reaction with NO, the emission of the rhodamine moiety in is switched off, which prevents the LRET process, so that the probe emits only the long-lived Tb3+ luminescence. However, upon reaction with NO, accompanied by the turn-on of rhodamine emission, the LRET from the Tb3+-complex moiety to rhodamine moiety occurs, which results in a remarkable increase of the rhodamine emission and decrease of the Tb3+ emission. After the reaction, the intensity ratio of the rhodamine emission to the Tb3+ emission, I565/I540, was found to be 28.8-fold increased, and the dose-dependent enhancement of the I565/I540 value showed a good linearity upon the increase of NO concentration. In addition, a dose-dependent luminescence lifetime decrease was distinctly observed between the average luminescence lifetime of the probe and NO concentration, which provides a ∼10-fold contrast window for the detection of NO. These unique properties allowed to be conveniently used as a time-gated luminescence probe for the quantitative detection of NO using both luminescence intensity ratio and luminescence lifetime as signals. The applicability of for ratiometric time-gated luminescence imaging of NO in living cells was investigated. Meanwhile, dye co-localization studies confirmed a quite precise distribution of in lysosomes by confocal microscopy imaging. Furthermore, the practical applicability of was demonstrated by the visualization of NO in Daphnia magna. All of the results demonstrated that could serve as a useful tool for exploiting and elucidating the function of NO at sub-cellular levels with high specificity, accuracy and contrast.

No MeSH data available.


(A) Steady-state emission spectra (λex = 330 nm) of free CTMR (1.0 μM, solid line) and TRP-Tb3+ (1.0 μM, dashed line). (B) Time-gated emission spectra (λex = 330 nm) of TRP-NO before (solid line) and after (dotted line) reaction with NO (CTRP-NO = 15 μM, CNO = 0.1 mM, incubation time: 50 min). The solvent was 0.05 M PBS buffer of pH 7.4.
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fig1: (A) Steady-state emission spectra (λex = 330 nm) of free CTMR (1.0 μM, solid line) and TRP-Tb3+ (1.0 μM, dashed line). (B) Time-gated emission spectra (λex = 330 nm) of TRP-NO before (solid line) and after (dotted line) reaction with NO (CTRP-NO = 15 μM, CNO = 0.1 mM, incubation time: 50 min). The solvent was 0.05 M PBS buffer of pH 7.4.

Mentions: To investigate the luminescence property of TRP-Tb3+, the steady-state emission spectra of free CTMR and TRP-Tb3+ excited at 330 nm in 0.05 M PBS buffer of pH 7.4 was measured. As shown in Fig. 1A, the Tb3+ emission is completely covered by the emission of CTMR moiety in the emission spectrum of TRP-Tb3+, and the emission intensity of TRP-Tb3+ at 580 nm (the maximum emission wavelength of the CTMR moiety) is 16.4-fold higher than that of free CTMR at the same concentration. By measuring the lifetime of the donor's emission in the absence of acceptor (τD, 1.1 ms)30 and that of the LRET-induced emission of the acceptor (τAD, 0.016 ms), the LRET efficiency (E) in TRP-Tb3+ was calculated to be 98.5%, using the equation31 of E = 1 – (τAD/τD), which reveals that the energy transfer from LTC to CTMR in the constructed molecular platform, TRP-Tb3+, is highly efficient. Fig. 1B shows the time-gated emission spectra of TRP-NO before and after reaction with NO in 0.05 M PBS buffer of pH 7.4. In the absence of NO, since the spirolactam derivative of CTMR moiety is non-fluorescent, TRP-NO emits only the Tb3+ luminescence. After being reacted with NO, accompanied by the opening of the spirolactam ring of CTMR moiety, the intramolecular LRET from LTC to CTMR is recovered, so that a dramatic enhancement of rhodamine emission and a remarkable decline of Tb3+ emission in the time-gated luminescence emission spectra are observed. Thus it is reasonable to conclude that TRP-NO could act as a ratiometric probe for the time-gated luminescence detection of NO with the intensity ratio of rhodamine emission to Tb3+ emission as the signal.


Development of a novel lysosome-targetable time-gated luminescence probe for ratiometric and luminescence lifetime detection of nitric oxide in vivo † † Electronic supplementary information (ESI) available: Experimental details for the syntheses of TRP-Tb 3+ and TRP-NO , and supplementary figures. See DOI: 10.1039/c6sc03667h Click here for additional data file.
(A) Steady-state emission spectra (λex = 330 nm) of free CTMR (1.0 μM, solid line) and TRP-Tb3+ (1.0 μM, dashed line). (B) Time-gated emission spectra (λex = 330 nm) of TRP-NO before (solid line) and after (dotted line) reaction with NO (CTRP-NO = 15 μM, CNO = 0.1 mM, incubation time: 50 min). The solvent was 0.05 M PBS buffer of pH 7.4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384565&req=5

fig1: (A) Steady-state emission spectra (λex = 330 nm) of free CTMR (1.0 μM, solid line) and TRP-Tb3+ (1.0 μM, dashed line). (B) Time-gated emission spectra (λex = 330 nm) of TRP-NO before (solid line) and after (dotted line) reaction with NO (CTRP-NO = 15 μM, CNO = 0.1 mM, incubation time: 50 min). The solvent was 0.05 M PBS buffer of pH 7.4.
Mentions: To investigate the luminescence property of TRP-Tb3+, the steady-state emission spectra of free CTMR and TRP-Tb3+ excited at 330 nm in 0.05 M PBS buffer of pH 7.4 was measured. As shown in Fig. 1A, the Tb3+ emission is completely covered by the emission of CTMR moiety in the emission spectrum of TRP-Tb3+, and the emission intensity of TRP-Tb3+ at 580 nm (the maximum emission wavelength of the CTMR moiety) is 16.4-fold higher than that of free CTMR at the same concentration. By measuring the lifetime of the donor's emission in the absence of acceptor (τD, 1.1 ms)30 and that of the LRET-induced emission of the acceptor (τAD, 0.016 ms), the LRET efficiency (E) in TRP-Tb3+ was calculated to be 98.5%, using the equation31 of E = 1 – (τAD/τD), which reveals that the energy transfer from LTC to CTMR in the constructed molecular platform, TRP-Tb3+, is highly efficient. Fig. 1B shows the time-gated emission spectra of TRP-NO before and after reaction with NO in 0.05 M PBS buffer of pH 7.4. In the absence of NO, since the spirolactam derivative of CTMR moiety is non-fluorescent, TRP-NO emits only the Tb3+ luminescence. After being reacted with NO, accompanied by the opening of the spirolactam ring of CTMR moiety, the intramolecular LRET from LTC to CTMR is recovered, so that a dramatic enhancement of rhodamine emission and a remarkable decline of Tb3+ emission in the time-gated luminescence emission spectra are observed. Thus it is reasonable to conclude that TRP-NO could act as a ratiometric probe for the time-gated luminescence detection of NO with the intensity ratio of rhodamine emission to Tb3+ emission as the signal.

View Article: PubMed Central - PubMed

ABSTRACT

Trp-notrp-notrp-notrp-notrp-notrp-notrp-no: Rapid, multiplexed, sensitive and specific identification and quantitative detection of nitric oxide (NO) are in great demand in biomedical science. Herein, a novel multifunctional probe based on the intramolecular LRET (luminescence resonance energy transfer) strategy, , was designed for the highly sensitive and selective ratiometric and luminescence lifetime detection of lysosomal NO. Before reaction with NO, the emission of the rhodamine moiety in is switched off, which prevents the LRET process, so that the probe emits only the long-lived Tb3+ luminescence. However, upon reaction with NO, accompanied by the turn-on of rhodamine emission, the LRET from the Tb3+-complex moiety to rhodamine moiety occurs, which results in a remarkable increase of the rhodamine emission and decrease of the Tb3+ emission. After the reaction, the intensity ratio of the rhodamine emission to the Tb3+ emission, I565/I540, was found to be 28.8-fold increased, and the dose-dependent enhancement of the I565/I540 value showed a good linearity upon the increase of NO concentration. In addition, a dose-dependent luminescence lifetime decrease was distinctly observed between the average luminescence lifetime of the probe and NO concentration, which provides a ∼10-fold contrast window for the detection of NO. These unique properties allowed to be conveniently used as a time-gated luminescence probe for the quantitative detection of NO using both luminescence intensity ratio and luminescence lifetime as signals. The applicability of for ratiometric time-gated luminescence imaging of NO in living cells was investigated. Meanwhile, dye co-localization studies confirmed a quite precise distribution of in lysosomes by confocal microscopy imaging. Furthermore, the practical applicability of was demonstrated by the visualization of NO in Daphnia magna. All of the results demonstrated that could serve as a useful tool for exploiting and elucidating the function of NO at sub-cellular levels with high specificity, accuracy and contrast.

No MeSH data available.