Fluorescence detection of target DNA-T utilizing the nucleic
2 2 2
acid probes was carried out in a H DCFDA–H O solution at
room temperature. The results are shown in Fig. S3 (see ESIw
for details). The addition of control sequence DNA-R shows
no significant enhancement in the fluorescence intensity (curve
b), whereas DNA-T enhances the fluorescence intensity more
than 3-fold compared to DNA-R. These observations suggests
that the probes are able to detect DNA-T effectively and
sensitively with respect to the fluorescence changes.
A calibration curve was established for the detection of
ꢀ
1
target DNA-T in a tris-HCl buffer containing 150 mmol L
ꢀ1
NH Ac and 20 mmol L KCl under optimal conditions. As
4
shown in Fig. 3, the fluorescence intensity is linearly
proportional to the concentration of DNA-T in the range of
ꢀ
ꢀ7
1
Fig. 2 Effect of KCl concentration on DF/F in 50 mmol L Tris-HCl
R
ꢀ1
ꢀ1
,
containing 150 mmol L NH
4
Ac (pH 7.0) with probes 5 ꢁ 10 mol L
.0 ꢁ 10 to 5.0 ꢁ 10ꢀ7 mol L . A linear regression eqn (1)
ꢀ8
ꢀ1
5
DNA-T 3.0 ꢁ 10ꢀ mol L and hemin 5.0 ꢁ 10 mol L
7
ꢀ1
ꢀ7
ꢀ1
.
was set up:
environment reduced the catalytic activity of the DNAzyme.
The results indicate that pH = 7.0 is the optimum condition
for this study.
F = 292.32 + 136.03C
(R = 0.9972)
(1)
DNAꢀT
where F is the fluorescence intensity at 525 nm, CDNAꢀT
represents the concentration of DNA-T, and R is the
regression coefficient. The detection limit is estimated to be
It is known that potassium ions show a higher affinity for the
G-quadruplex. Nevertheless, the DNAzymes (G-quadruplex–
hemin complex) in this study are prepared using ammonium
ꢀ
9
ꢀ1
7.0 ꢁ 10 mol L (defined as three times the concentration
corresponding to the standard deviation of the blank).
1
8
cations, which possess higher catalytic activity. The effect of
KCl concentration in buffer on DF/F was therefore studied.
As shown in Fig. 2, the enhancement rate increases with
increasing KCl concentration. When the KCl concentration
R
In summary, a novel and simple sensor using binary probes
for detection of GMO with good selectivity and sensitivity was
successfully developed. The sensor is able to fold into a
G-quadruplex structure in the presence of DNA-T and give a
G-quadruplex–hemin complex via complexation with hemin.
ꢀ
1
is higher than 20 mmol L , DF/F
results are in agreement with the findings reported by Sen
R
decreases remarkably. The
2
1
ꢀ1
et al. In this study, 20 mmol L of KCl was used as the
optimized concentration for GMO detection.
This unique complex was found to effectively catalyze H
O
2 2
oxidation of H DCFDA, and thus turned on fluorescence in
2
Although the complex produced from hemin and
GMO detection. Based on the changes of fluorescence
intensity, the DNA-T is able to quantify in the range of
22
G-quadruplex has a superior catalytic activity, the unreacted
20
ꢀ8
ꢀ7
ꢀ1
hemin in solution causes background noise. Hence, the effect of
hemin concentration is also an important factor for investigation.
Fig. S2 (see ESIw for details) shows the relationship between
5.0 ꢁ 10 to 5.0 ꢁ 10 mol L with a detection limit of
ꢀ9 ꢀ1
7.0 ꢁ 10 mol L
.
The authors gratefully acknowledge the financial support of
the NSFC (20735002, 40940026 and 20905013), the National
Basic Research Program of China (No. 2010CB732403),
and the Special Foundation for Young Scientists of Fujian
Province, China (2007F3081, 2008F3057).
DF/F
R
R
and hemin concentration. The highest DF/F value was
ꢀ
7
ꢀ1
.
obtained when the hemin concentration was 1.0 ꢁ 10 mol L
Therefore, the optimized concentration of hemin was found to
be 1.0 ꢁ 10ꢀ mol L
7
ꢀ1
.
Notes and references
1
L. T. Yang, A. H. Pan, J. W. Jia, J. Y. Ding, J. X. Chen, H. Cheng,
C. M. Zhang and D. B. Zhang, J. Agric. Food Chem., 2005, 53,
1
83–190.
H.-Y. Huang and T.-M. Pan, J. Agric. Food Chem., 2004, 52,
264–3268.
T. W. Alexander, T. Reuter and T. A. Mcallister, J. Agric. Food
Chem., 2007, 55, 2918–2922.
J. K. RHO, T. Lee, S.-I. Jung, T.-S. Kim, Y.-H. Park and
Y.-M. Kim, J. Agric. Food Chem., 2004, 52, 3269–3274.
T. Watanabe, S. Tokishita, F. Spiegelhalter, S. Furul, K. Kitta,
A. Hino, R. Matsuda, H. Akiyama and T. Maitani, J. Agric. Food
Chem., 2007, 55, 1274–1279.
2
3
4
5
3
6
European Commission Regulation (EC) No. 1829/2003 and 1830/
2003, Off. J. Eur. Commun. L268, 128 (Oct 18, 2003).
ꢀ
1
Fig. 3 Calibration curve for DNA-T in 50 mmol L
Tris-HCl
ꢀ
1
ꢀ1
ꢀ7
containing 150 mmol L NH
4
ꢀ1
Ac and 20 mmol L KCl (pH 7.0)
7 Notification No. 2000-31. Ministry of Agriculture and Forestry of
Korea, Seoul, Korea (Apr 22, 2000).
8 Notification No. 1775 Food and Marketing Bureau, Ministry of
Agriculture, Forestry and Fisheries of Japan, Tokyo, Japan
ꢀ7
ꢀ1
with probes 5 ꢁ 10 mol L and hemin 1.0 ꢁ 10 mol L . A:
ꢀ1
fluorescence curve with various concentration (mol L ), (a) 0.0, (b)
ꢀ7
ꢀ8
ꢀ7
ꢀ7
ꢀ7
5
.0 ꢁ 10 , (c) 1.0 ꢁ 10 , (d) 2.0 ꢁ 10 , (e) 3.0 ꢁ 10 , (f) 4.0 ꢁ 10
,
(Jun 10, 2000).
W.-T. Xu, K.-L. Huang, Y. Wang, H.-X. Zhang and Y.-B. Luo,
J. Sci. Food Agric., 2006, 86, 1103–1109.
ꢀ7
(
g) 5.0 ꢁ 10 . B: linear relationship between fluorescence intensity and
9
DNA-T concentration. The PMT detector voltage is 700 V.
1
438 Chem. Commun., 2011, 47, 1437–1439
This journal is c The Royal Society of Chemistry 2011