A novel aptasensor for the ultra-sensitive detection
of adenosine triphosphate via aptamer/quantum dot
based resonance energy transfer†
Zheng Li,‡
ab
Yijing Wang,‡
a
Ying Liu,
b
Yongyi Zeng,
d
Aimin Huang,
ac
Niancai Peng,
b
Xiaolong Liu
*
ac
and Jingfeng Liu
*
ac
We designed a novel aptamer based biosensor (aptasensor) for
ultrasensitive detection of adenosine triphosphate (ATP) through
resonance energy transfer (RET). The A TP aptamer was modified with
Cy3 at the 3
0
end, and a green quantum dot (525) was attached to the
5
0
end of its complementary sequence respectively. The ATP aptamer
and its complementary sequence could assemble into a duplex
structure in the absence of target ATP, and then decrease the distance
between the quantum dot and Cy3 which could produce significant
RET signal. Upon ATP binding, the ATP aptamer could dissociate with
its complementary sequence and then increase the distance between
the quantum dot and Cy3 which would significantly decrease the RET
signal. Therefore, the ATP detection could be easily achieved through
detection of the fluorescence intensity ratio between 525 nm and
560 nm. The results show that the emission fluorescence intensity
ratio of 525/560 is linearly related to the logarithmic concentration
of ATP. The linear range of this aptasensor is from 0.1 nM to 1 mM,
and the detection limit is lower down to 0.01 nM. Excellent selectivity
of this aptasensor for ATP has been demonstrated through the
detection of thymidine triphosphate (TTP), cytidine triphosphate
(CTP), guanosine triphosphate (GTP) and adenosine diphosphate
(ADP) respectively as control. The method we described here could
easily detect ATP with excellent selectivity, linearity and sensitivity
down to the nanomolar range, as well as avoid photobleaching.
Aptamers are in vitro selected functional single-stranded DNAs
(ssDNAs), RNAs, or even chemically modied nucleic acids
1
which could fold into special structures and possess high
recognition ability to specic targets ranging from metal ions,
2
organic and inorganic small molecules,
3
proteins,
4
and even
whole cells.
5
They have been extensively used to construct
different biosensor systems and different detection strategies.
6
Adenosine 5
0
-triphosphate (ATP) is the major energy source
in living organisms. The development of a fast, sensitive and
quantitative ATP detection strategy would have great impor-
tance in both basic studies as well as in the diagnostic eld. ATP
is usually detected by methods based on chromatography,
bioluminescence or chemiluminescence.
7
However, the chro-
matography based methods suffer from the tedious separation
of sample and low accuracy of detection. Although both
chemiluminescence and bioluminescence based methods
have good sensitivity, the stability and specicity are always a
problem. High stability, specicity and sensitivity would always
be the challenges in the ATP detection strategy development.
So far, various designs of aptamer based biosensors (apta-
sensor) for ATP detection have been developed based on uo-
rescence,
8
electrochemical,
9
and colorimetric detection.
10
Compared to other methods, uorescent aptasensors are
particularly interesting due to their high stability, high sensitivity
and feasibility of quantication. Quantum dots (QDs) have the
advantages of broad absorption with narrow emission spectra,
high quantum yield, high chemical stability and resistance to
photo-bleaching.
11
Recently, several groups have reported
aptamer/QD based biosensors for ATP detection.
12
However, they
still suffer from complicated preparation and purication
procedures, as well as low sensitivity. In this paper, we developed
a fast and simple aptamer/QD based biosensor for ultrasensitive
ATP detection based on resonance energy transfer (RET).
The design principle for the RET detection of ATP is illustrated
in Scheme 1. Here, the ATP aptamer (5
0
-ACCTGGGGGAGTATT
GCGGAGGAAGGT) was synthesized and its 3
0
-end was labeled
with Cy3 (Shanghai shenggong, China). The comp lementary
sequence of the ATP aptamer was modied with an amino group
at its 5
0
end through a (CH
2
)
12
linker (3
0
-TGGACCCCCTCATAA
CGCCTCCTTCCA–(CH
2
)
12
–NH
2
, Shanghai Shenggong, China).
a
Mengchao Hepatob iliary Hospital of Fujian Medical University, Fuzhou 350025,
People's Republic of China. E-mail: xiaoloong.liu@gmail.com
b
State Key Laboratory for Manufacturing System Engineering, School of Mechanical
Engineering, Xi'an Jiaotong University, Xi'an 710049, People's Republic of China
c
The Liver Center of Fujian Province, Fujian Medical University, Fuzhou 350025,
People's Republic of China
d
Liver Disease Center, The First Affiliated Hospital of Fujian Medical University,
Fuzhou 350007, People's Republic of China
† Electronic supplementary inform ation (ESI) available. See DOI:
10.1039/c3an00449j
‡ Equal contribution authorship.
Cite this: Analyst, 2013, 138, 4732
Received 5th March 2013
Accepted 8th June 2013
DOI: 10.1039/c3an00449j
www.rsc.org/analyst
4732 | Analyst, 2013, 138, 4732–4736 This journal is ª The Royal Society of Chemistry 2013
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