Abstract
This protocol describes the synthesis of modular turn-ON QCy7-based probes for the detection of biologically relevant analytes, such as hydrogen peroxide, ubiquitous sulfhydryl and β-galactosidase. The probes presented herein are prepared through a simple procedure that involves the preliminary alkylation of 4-hydroxy-isophthalaldehyde with a relevant analyte-responsive protecting group, followed by condensation of the resulting product with 2 equivalents of sulfo-indolium moieties. Evaluation of the turn-ON near-IR fluorescence response to their relevant analytes for the three different QCy7 probes is also reported. The preparation of a QCy7 diagnostic probe requires 1–2 d. Probes for other analytes can be prepared according to this modular procedure by incorporating a specific analyte-responsive group as a triggering substrate.
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Acknowledgements
D.S. thanks the Israel Science Foundation (ISF), the Binational Science Foundation (BSF) and the German-Israeli Foundation (GIF) for financial support. This work was partially supported by grants from the Israeli National Nanotechnology Initiative (INNI), Focal Technology Area (FTA) program: Nanomedicine for Personalized Theranostics; and by The Leona M. and Harry B. Helmsley Nanotechnology Research Fund.
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O.R., E.K.-F., S.F., R.S.-F. and D.S. designed the experiments; O.R., E.K.-F. and S.F. conducted the experiments; O.R., E.K.-F., S.F., R.S.-F. and D.S. analyzed the data; and O.R., E.K.-F., S.F., R.S.-F. and D.S. wrote the manuscript.
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A patent related to this work has been filed by Tel Aviv University (reference no. PCT/IB2012/051255). The title of the invention is: 'Activatable Fluorogenic Compounds And Uses Thereof As Near-Infrared Probes'.
Integrated supplementary information
Supplementary Figure 6 Fluorescence and absorption spectra of protected and unprotected QCy7.
Fluorescence a) and absorption b) spectra of protected (red) and unprotected (green) QCy7, [100 μM], 1%DMSO in 0.1 M PBS, pH=7.4, λex=590 nm, λem=720 nm.
Supplementary Figure 7 NIR fluorescence spectra of probe 1 in the presence or absence of H2O2.
a) NIR Fluorescence (λex=590 nm, λem=720 nm) emitted upon incubation of probe 1 [100 μM], in the presence (green) or absence (red) of H2O2 (5 equivalents) in 1%DMSO in 0.1 M PBS, pH=7.4. b) NIR Fluorescence (λex=590 nm, λem=720 nm) emitted upon incubation of probe 1 [100 μM] with 0, 10, 20, 30, 40, 70 and 100 μM H2O2, in 1%DMSO in 0.1 M PBS, pH=7.4.
Supplementary Figure 8 NIR fluorescence spectra of probe 2 in the presence or absence of cysteine.
a) NIR Fluorescence (λex=590 nm, λem=720 nm) emitted upon incubation of probe 2 [100 μM], in the presence (green) or absence (red) of cysteine (1 equivalent) in 1%DMSO in 0.1 M PBS, pH=7.4. b) NIR Fluorescence (λex=590 nm, λem=720 nm) emitted upon incubation of probe 2 [100 μM] with 0.1, 2.5, 5, 10, 15, 20 μM cysteine, in 1%DMSO in 0.1 M PBS, pH=7.4.
Supplementary Figure 9 NIR fluorescence spectra of probe 3 in the presence or absence of β-galactosidase.
a) NIR Fluorescence (λex=590 nm, λem=720 nm) emitted upon incubation of probe 3 [100 μM], in the presence (green) or absence (red) of β-galactosidase (1.37 units) in 1%DMSO in 0.1 M PBS, pH=7.4. b) NIR Fluorescence (λex=590 nm, λem=720 nm) emitted upon incubation of probe 3 [100 μM] with 1.37, 0.91, 0.45, 0.22, 0.11 or 0 enzyme units of β-galactosidase, in 1%DMSO in 0.1 M PBS, pH=7.4.
Supplementary information
Supplementary Figure 1
Activation mechanism of QCy7-based probe 1 by H2O2 to produce a fluorescent turn-ON response. (PDF 120 kb)
Supplementary Figure 2
Activation mechanism of a QCy7-based probe 2 by a ubiquitous thiol to produce a fluorescent turn-ON response. (PDF 93 kb)
Supplementary Figure 3
Activation mechanism of a QCy7-based probe 3 by β-galactosidase to produce a fluorescent turn-ON response. (PDF 106 kb)
Supplementary Figure 4
Synthesis of intermediate 2a. (PDF 141 kb)
Supplementary Figure 5
Synthesis of intermediate 3a. (PDF 138 kb)
Supplementary Figure 6
Fluorescence and absorption spectra of protected and unprotected QCy7. (PDF 206 kb)
Supplementary Figure 7
NIR fluorescence spectra of probe 1 in the presence or absence of H2O2. (PDF 254 kb)
Supplementary Figure 8
NIR fluorescence spectra of probe 2 in the presence or absence of cysteine. (PDF 249 kb)
Supplementary Figure 9
NIR fluorescence spectra of probe 3 in the presence or absence of β-galactosidase. (PDF 248 kb)
Supplementary Methods
Supplementary Methods (PDF 296 kb)
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Redy-Keisar, O., Kisin-Finfer, E., Ferber, S. et al. Synthesis and use of QCy7-derived modular probes for the detection and imaging of biologically relevant analytes. Nat Protoc 9, 27–36 (2014). https://doi.org/10.1038/nprot.2013.166
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DOI: https://doi.org/10.1038/nprot.2013.166
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