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it is conceivable that when excessive amount of Cys exists in the
reaction media, a Cys–Hcy mixed disulfide could also form,
leading to an increase of the ‘‘effective’’ concentration of Hcy.
This explains why Cys alone does not trigger fluorescence
increase but causes a B40% increase in fluorescence response
to Hcy. It should be noted that a previously published dansyl azide
analogue, 1,5-DNS-Az also shows selectivity for Hcy (Fig. S6, ESI†).
In addition, the influence of pH on the selectivity has also
been tested using 96-well plate and a microplate reader (Fig. S7,
ESI†). Sodium phosphate buffer (100 mM) in the pH range of
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5.3–11.5 was used in the experiments. A pH dependent fluores-
2
cence response was observed for DN-2 in the presence of thiols.
It was found that DN-2 itself remains fluorescently stable at pH
values lower than 10.5. The selectivity for Hcy increased with
increasing pH from 5–7.4. In buffers at pH higher than 7.4,
reactions with Cys and GSH were promoted. Therefore, the
highest selectivity was observed at pH 7.4, which is the physio-
logical pH. We have also examined the reaction time profile in
phosphate buffer at 37 1C (Fig. S8, ESI†). The result has
suggested that heat facilitated the reaction. The reaction time
for 120 mM of DN-2 and 100 mM of Hcy decreased from 60 min
at room temperature (about 22 1C) to 30 min at 37 1C.
Hcy is a very important biomarker for the diagnosis and
prognosis of various diseases. However, due to the structural
similarity of Hcy and Cys, direct detection of this biomarker
still remains a major challenge. We have developed a novel
redox sensitive fluorescent probe for quantitative Hcy analysis.
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estimation of Hcy concentration. This probe will be very useful
for the selective detection of Hcy in complex biological samples.
We are grateful for the financial support from Center for
Diagnostics and Therapeutics for a CDT/University fellowship
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Chem. Commun., 2014, 50, 13668--13671 | 13671