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ChemComm
Page 4 of 4
DOI: 10.1039/C6CC08657H
COMMUNICATION
Journal Name
The level of glycosaminoglycans (GAGs) modifying enzymes in switching at physiological pH in water. The heparin induced
human plasma is an important bio‐marker of the progression reversible aggregate formation was further utilized in
of many lethal diseases including cancer. For example, an developing a new easy‐to‐operate assay for heparinase I in
enhancement in heparinase activity is considered to be an biological fluids. The dose dependent emission modulation
indication of multiple myeloma.12 However, fluorometric was also observed on low‐cost color strips, which ensured
assay for heparinase is really scarce in litereture.13 Therefore, rapid onsite detection of heparin in unknown samples.
we were interested to employ the heparin mediated reversible
aggregation of our probe molecules for fluorometric
estimation of heparinase I. A drastic quenching in the
aggregate emission (at 600 nm) was observed upon incubation
Notes and references
1
2
R. J. Linhardt and T. Toida, Acc. Chem. Res., 2004, 37, 431.
I. Caplia and R. J. Linhardt, Angew. Chem. Int. Ed., 2002, 41
390.
,
of the pre‐formed 1+heparin conjugate with heparinase I for
~30 min (Fig S15, ESI†). The degradation process was observed
to follow a 1st order kinetics in the presence of excess of
enzyme (Fig S16a, ESI†). The Michalis‐Menten constant was
determined to be 2.32 x 10‐6 M for heparinase I following the
present protocol, which appeared to be in good agreement
with the literature (Fig 5b & S16b, ESI†).14 To ensure the
specificity of this newly developed protocol the enzymatic
3
4
G. J. Despotis, G. Gravlee, K. Filos and J. Levy, Anesthesiology,
1999, 91, 1122.
(a) B. Boneu and P. Moerloose, Semin. Thromb. Hemost.,
2001, 27, 519. (b) D. J. Murray, W. J. Brosnahan, B. Pennell,
D. Kapalanski, J. M. Weiler and J. Olson, J. Cardiothorac.
Vasc. Anesth., 1997, 11, 24.
5
(a) Z. Zhenlin and E. V. Anslyn, J. Am. Chem. Soc., 2002, 124,
9014. (b) M. S. Bromfield, E. Wilde, D. K. Smith, Chem. Soc.
Rev., 2013, 42, 9184 and all the references there in. (c) C. W.
Chan and D. K. Smith, Chem. Commun., 2016, 52, 3785. (d) R.
assay was also performed in the presence of normal
‐
glucosidase. However, in this case no spectral change was
identified even after 20 min of incubation at pH 7.4 (Fig 5c).
Further heparinase estimation was also achieved in diluted
serum (5%) medium. Here also a decrease in aggregated
S. Dey and C. R. Raj, Chem. Asian J., 2012, 7, 417.
6
7
(a) K.‐Y. Pu and B. Liu, Macromolecules, 2008, 41, 6636. (b) S.
L. Wang and Y. T. Chang, Chem. Commun., 2008, 1173. (c) Q.
Dai, W. Liu, X. Zhuang, J. Wu, H. Zhang and P. Wang, Anal.
Chem., 2011, 83, 6559.
emission was observed upon incubation of
conjugate with enzyme for requisite time (S17, ESI†).
1+heparin
(a) S. Bhattacharya and S. K. Samanta, Chem. Eur. J., 2012,
18, 16632. (b) S. K. Samanta and S. Bhattacharya, J. Mater.
Chem., 2012, 22, 25277.
(a) P. Job, Ann. Chim., 1928,
Am. Chem. Soc., 1949, 71, 2703.
W. Sun, H. Bandmann and T. Schrader, Chem. Eur. J., 2007,
13, 7701.
(a)
(b)
8
9
9, 113. (b) B. H. Hildebrand, J.
0
0.2 0.4 0.6 0.8 1.0
1
2
3
4
5
6
7
Cross‐reactivitystudies
Heparin
µg/mL
10 S. S. Babu, K. K. Kartha and A. Ajayaghosh, J. Phys. Chem.
Lett., 2010, , 3413.
Fig. 6 (a) Color changes of compound coated TLC plates upon dipping
into the solution of heparin of different concentration in water (pH
7.4) [under ~360 nm UV light]. (b) Color change (under ~360 nm UV
lamp) of compound coated TLC plates upon dipping into different
1
11 Y. T. Shen, C. H. Li, K.C. Chang, S. Y. Chin, H. A. Lin, Y. M. Liu,
C. Y. Hung, H.‐F. Hsu and S. S. Sun, Langmuir, 2009, 25, 8714.
12 R. Sasisekharan, Z. Shriver, G. Venkataraman and U.
analyte solution; No analyte [
1] and with Chs [
2], HA [3], Heparin [4],
Narayanasami, Nat Rev Cancer, 2002,
13 (a) Z. Ban, C. J. Bosques and R. Sasisekharan, Org. Biomol.
Chem., 2008, , 4290. (b) Y. Ding, L. Shi and H. Wei, Chem.
Sci., 2015, , 6361
2, 521.
Protamine [ ], Polylysine [ ], Chitosan [7
5
6
] (pH 7.4).
6
Further, the dose‐dependent changes in emission signal with
heparin encouraged us to develop easily obtainable solid‐
support based portable devices for on‐site heparin detection
and quantification.15 A gradual change in color from sky‐blue
to yellow (under long UV light) was observed upon dipping the
compound coated TLC plates into heparin solution of different
concentrations (Fig 6a). Control experiments were also
designed by dipping the strips into solution of HA and Chs to
verify the selectivity of the present protocol (Fig 6b). No
detectable color change in these two cases ensured the
specificity of the sensor towards heparin over the other
potential competitors. Thus, it could be easily concluded that
the present protocol assured the rapid on‐site identification of
heparin along with proper quantification.
6
14 V. C. Yang, R. J. Linhardt, H. Bernstein, C. L. Cooney, R.
Langer, J. Biol. Chem., 1985, 260, 1849.
15 (a) N. Kumari, N. Dey, K. Kumar and S. Bhattacharya, Chem.
Asian J., 2014, 9, 3174. (b) N. Dey, S. K. Samanta and S.
Bhattacharya, ACS Appl. Mater. Interfaces, 2013, 5, 8394.
Conclusions
In conclusion, we have introduced conjugated bis‐pyridinium
phenylenevinylene based molecular probes for their unique
heparin triggered dose‐dependent, multi‐color emission
4 | J. Name., 2012, 00, 1‐3
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