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The concentration of cAMP was also quantified from human
blood sample collected from P. M. Hospital, Visva-Bharati
University. 4 ml human blood of normal healthy person was
centrifuged to collect the blood cells. Blood cells were
dissolved with 10 mM Tris-Cl buffer (pH 7.0) and lysed by
osmotic shock with ice cold water. Supernatant were collected
after centrifugation and analyzed to detect cAMP present in
this sample with the help of cAMP standard fluorescence
curve (Fig.5c and S17) using the selective detection ability of
Laudette, C. Conte, Circ. Res. 201D6O,1I:1180.1039/C7CC02935G
, 881-897.
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5
6
(a) M. Klein, T. Bopp, Front Immunol. 2016, 7, 315.(b)
V. L. Wehbi, K. Taskén, Front. Immunol. 2016,
7, 222.
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(a) M. S. Shim, K. Y. Kim, W. K. Ju, BMB Rep. 2016,
3717. (b) L. Tetsi, A. L. Charles, S. Paradis, A. Lejay, S.
Talh,a, B. Geny, C. Lugnier, Cell Mol Life Sci. 2016, doi:
10.1007/s00018-016-2446-0 (c) O. Torres-Quesada, R.
the probe NpRD
.
All estimation has been done in
triplicate.From the standard curve it has been found that the
concenctration of cAMP in the trested blood samples are 2.8
7
M.To authenticate the above result the real sample was
spiked with known concentration of cAMP and used for
sensing purposes by adding known volumes from each sample
to the NpRD. The recovery of the spiked samples was
estimated to be in the range of 96–98% (Table S2, ESI†).We
further validated assay of cAMP from multiple human blood
samples using NpRD. Each sample has been assayed in
triplicate. The signal to noise ratio of each independent
reading is between 16 and 21 (Table S3). The fold increase of
fluorescence signals have also been statistically validated after
calculating the Z’ value for each set of measurement.19 It has
been found that in all tested samples the Z’ score is more than
Röck,
E. Stefan, Horm.
Metab.
Res.2016,
doi.org/10.1055/s-0042-110791.
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(a) Y. -K. Yang, H. J. Cho, J. Lee, I. Shin, J. Tae, Org. Lett.
2009, 11, 859. (b) Y. -K. Yang, S. Lee, J. Tae, Org. Lett.
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Chemistry and Biology I. Springer, New York, 2010,
8
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11 (a) H. S. Sarkar, S. Das, M. R. Uddin, S. Mandal, P.
Sahoo, Asian J. Org. Chem. 10.1002/ajoc.201600516 (b)
P. Sahoo, H. S. Sarkar, S. Das, K. Maiti, M. R. Uddin, S.
0.9, indicating an optimized and validated assay of cAMP
.
In conclusion, we have successfully developed an unique
naphthol-based rhodamine derivative NpRD for the selective
and differential detection of cAMP and other adenosine
phosphates in aqueous medium with very low concentration.
NpRD exhibits highly sensitive and selective response
differently towards cAMP and other APs both in the
colorimetric and fluorimetric detection technique. This is the
first known method where an anion sensing mechanism is
based on the change in structure from spiro cyclic to open
rhodamine form to make the probe fluorescent. The ‘turn on’
fluorescence for the selective detection of cAMP was
successfully demonstrated in live cell and in human blood
sample. This proves the potentiality of probe NpRD useful for
investigating the presence of cAMP in biological systems.Our
newly designed fluorescence chemosensor that specifically
binds cAMP with detection limit in micromolar range makes it
appropriate to use in diagnostics as well as in live cell imaging.
The simple detection of cAMP by this chemosensor only
through colorimetry or fluorometry makes it unique for
probable usage in high throughput screening for mankind over
existing biosensors which estimates cAMP indirectly.
Mandal, RSC Adv. 2016,
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PS acknowledges SERB-DST, Govt. of India for awarding her the
young scientist grant [Project file no. SB/FT/CS-021/2014].SD
thanks UGC, India for research fellowship. Authors are grateful
to DBT-IPLS facility, CU; Prof. G. K. Das, Visva-Bharati, India, for
assistance during DFT calculations and Suman K. Mandal, SNU,
Noida, India for crystal data refinements.
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K. Swamy, S. K. Kim, S. Kim, S. H. Lee, J. Yoon,
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Notes and references
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18 J. Ratha, K. A. Majumdar, S. K. Mandal, R. Bera, C.
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