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the commonly used method in clinical labs, our proposed
method is easy to operate and the detection limit is sufficient
for HSA detection in human urine. The quantification experiment
further confirmed that DH1 was accurate and effective for quanti-
tative detection of trace HSA in human urine (Table S1, ESI†).
In summary, we have reported a new dicyanomethylene-4H-
chromene based probe DH1 that showed great sensing pro-
perties for HSA. DH1 exhibited an obvious HSA induced large
fluorescence enhancement in emission spectra without inter-
Fig. 3 A stereoview of the aligned crystal structures of serum albumins.
(
a) HSA, blue (PDB code 2BXP); BSA, yellow (PDB code 4F5S); binding site II
with ibuprofen is shown by the transparent red ball; (b) HSA, blue (PDB code
2
BXG); BSA, yellow (PDB code 4F5S); binding site I with phenylbutazone is ference from different ions and other biomolecules that are
shown by the transparent red ball; and (c) detailed view of the aligned commonly found in the environment or biosystems. Different
binding site I shows that the Leu237 residue of BSA occupied site I of BSA.
fluorescence responses to HSA and BSA may provide us a simple
method to selectively discriminate the two similar proteins. The
The significantly different fluorescence responses of DH1 molecular docking method was for the first time used to display
toward HSA and BSA motivated us to find the internal cause. the interactions between DH1 and binding site I of HSA and
In an effort to identify the difference in 3D structures, the X-ray different sensing processes towards HSA and BSA. The practical
crystal structures of HSA compounded with phenylbutazone applications showed that DH1 can detect trace HSA in human
(PDB code 2BXP) and HSA compounded with ibuprofen (PDB urine. We expect this new probe to be useful for more chemical
code 2BXG) and BSA (PDB code 4F5S) were collected from the and medical applications.
PDB database (http://www.rcsb.org/pdb). Structural analysis
This work was supported by NSF of China (21136002,
demonstrated that site II of BSA was similar to that of HSA 20923006 and 21076032), National Basic Research Program of
(Fig. 3a). However, site I of BSA was occupied by the Leu237 China (2013CB733702) and National High Technology Research
residue compared to the hollow site I of HSA (Fig. 3b and c). and Development Program of China (863 Program, 2011AA02A105).
Therefore we speculated that probe DH1 selectively binds to
site I of HSA.
Notes and references
To confirm that the additional Leu237 in site I of BSA
1
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hampered the insertion of the probe, DH1 was docked with
the drug binding site I in 4F5S, using the LigandFit module in
Discovery Studio 2.5. The docking simulation results showed
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into site I of HSA (Fig. S6b, ESI†), in which hydrogen bonds play
an important role in the protein–ligand interactions and make
a great contribution to the binding affinity. In the crystal
structure of HSA (2BXP), there was one hydrogen bond between
the carbonyl group of phenylbutazone and the guanidine group
of Arg218 (Fig. S6a, ESI†). However the cyano group and oxygen
atom of DH1 formed two hydrogen bonds with the guanidine
groups of Arg218 and Arg257 separately (Fig. S6b, ESI†). These
docking results demonstrated that DH1 selectively binds to site
I of HSA, which resulted in the difference in the fluorescence
response from BSA.
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HSA reached 0.19 mg mL
(Fig. S7, ESI†). Human urine
containing HSA at different concentrations was also prepared.
These urine solutions were then incubated with 5 mM probe
DH1. Fluorescence signals were measured and a good linear
relationship was obtained in this solution with a detection limit
3s/slope) of 5.51 mg L (Fig. S8, ESI†). Compared with RIA,
À1
(
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Chem. Commun., 2014, 50, 9573--9576 | 9575