With the increasing requirements on the sensitivity of biosensing system, various
strategies have been devised to boost detection sensitivity of chemosensing and
biosensing processes via the enhancement/amplification of sensing responses. The
recognition element and the signal transducer are two core sections in a biosensing
system. Various recognition elements have been developed in the past several decades,
such as enzymes, lectins, antibodies, molecular imprinting and aptamers. Aptamer,
which is single-stranded oligonucleotides with high affinity to a special target, is one
of the emerging classes of versatile receptors. This study aims to the design and
application of signal enhancement strategies in DNA aptamer-based biosensing. The
main content is as follows:
1. Because of its unique optical properties especially high extinction coefficient
(3-5 orders of magnitude higher than that of organic chromophores), gold nanoparticle
(AuNP) was employed as the signal enhancement element and transducer to construct
biosensing system for lead (II) detection. The sensing systems with two guanine-rich
sequences (TBA (5'-GGT TGG TGT GGT TGG-3') and PW17 (5'-GGG TAG GGC GGG TTG GG-3')) respectively as recognition elements were developed base on the
principle that ummodified AuNP can distinguish unfolded ssDNA from folded ssDNA,
such as G-quadruplex. The formation of G-quadruplexes by TBA-Pb2+ and
PW17-Pb2+ were characterized by circular dichroism. The specificity of the sequence
to Pb2+ was analyzed by difference absorption spectrum. The AuNP of 13 nm
synthesized using citrate reduction method was characterized by TEM and UV-vis
spectroscopy. Experimental conditions, such as NaCl concentrations, ssDNA
concentrations, aggregation time after the addition of salt were optimized. Results
showed that a limit of detection of 30 nM can be easily obtained for Pb2+ detection.
The PW17 system was found to possess a much better performance for Pb2+ detection
than TBA system. In the same [Pb2+] range and ssDNA concentration, PW17 system
shows a larger LSPR response than TBA system with a relative smaller standard
deviation.
2. To investigate the difference of TBA system and PW17 system in the
performance of Pb2+ sensing, the interaction between DNA G-quadruplexes and 13
nm gold nanoparticles (AuNPs) was studied. The adsorption of DNAs in
G-quadruplex solutions onto AuNPs was observed in DNA - AuNP-based sensing
system. The adsorption behavior was studied through monitoring of the localized
surface plasmon resonance (LSPR) absorbance of 13 nm AuNPs at 520 and 650 nm
(A650/A520) in the solutions of three widely studied guanine-rich sequences, TBA,
PW17, and PSO (5'-GGG TTA GGG TTA GGG TTA GGG-3'). It was found that the
degree of the adsorption of DNAs in Pb2+ stabilized G-quadruplex solutions is up to
93% after more than 5 h of incubation. Two interpretations, the adsorption of
G-quadruplexes and the unfolding of G-quadruplexes in the presence of AuNPs, were
proposed for these observations. To explore the possible explanation, the lead
concentrations in the solutions containing G-quadruplex and AuNP were analyzed by
inductively coupled plasma atomic emission spectrometer. The results showed that
Pb2+ had been released from the G-quadruplexes, which means the G-quadruplexes
may be unfolded in the presence of AuNPs. The adsorption rate in PW17-Pb2+ system
was lower than that in TBA-Pb2+ system, demonstrating that the G-quadruplex formed
from PW17 and Pb2+ is more stable in the presence of AuNPs. This result can
interpret the difference in their performance in Pb2+ sensing. Similar results were also
observed in PSO-K+ system, which indicate that the potential unfolding of
G-quadruplexes in the presence of AuNPs is a general phenomenon in DNA -
AuNP-based sensing system. This interaction between G-quadruplexes and AuNP
demonstrated that long time incubation between DNAs and AuNPs would possibly
make it unable to distinguish G-quadruplex from ssDNA.
3. To avoid the adverse effect of unmodified AuNPs on the formation of folded
structure of ssDNA, the thiolated-aptamer conjugated AuNPs sensing system was
adopted for biosensors design. The newly designed AuNP functionalized with split
aptamer was developed for the detection of adenosine triphosphate (ATP). The ATP
aptamer was split into two parts with their 5' prime or 3' prime modified with thiol.
Both the 5' SH and 3' SH modified strands for each split aptamer fragment were functionalized onto the same AuNP to construct double-functionalized AuNP-DNA
conjugates. Thus, the split aptamer can be reassembled into intact folded structure in
the presence of ATP molecule with two potential assembly types, which induces the
assembly of AuNP-DNA conjugates. In this double-functionalized system, the
traditional assembly type might facilitate another assembly type, which was found to
give two-fold increase in LSPR response of AuNPs in the presence of ATP than the
traditional assembly type, and improved the sensitivity for ATP detection. Time
courses of the assemble processes with different assembly types, Mg2+ concentrations,
and aptamer fragments densities on AuNP were followed using the absorption ratio at
650 nm and 520 nm. A limit of detection of 24 M with highly selectivity was
determined which has greatly surpassed the traditional assembly type in ATP sensing.
4. However, the double-functionalized AuNP-DNA system could only provide
two-fold increase in LSPR response of AuNPs to target molecules than the traditional
one. Therefore, a strand displacement reaction (SDR)-based catalytic cycle was
employed to amplify signals. This system involves an entropy-driven catalytic cycle
of two strand displacement reactions with five oligonucleotides, denoted as
Substrate-1", "Fuel-1", "Catalyst-1", "C1" and "C2", respectively. The "Catalyst-1
is an ATP aptamer catalyzing the SDRs to form the "Substrate-Fuel-1" duplexes. All
the intermediates in the SDR processes have been identified by PAGE
(polyacrylamide gel electrophoresis) analysis. Introduction of ATP into the SDR
system will induce the "Catalyst-1" to form G-quadruplex conformation so as to
inhibit the catalytic activity and cut down the formation of the "Substrate-Fuel-1
duplexes. Obviously, this target-inhibited catalytic cycle can be applied to an ATP
sensing system. When the "Substrate-1" and "Fuel-1" oligonucleotides were labeled
with a carboxyfluorescein (FAM) fluorophore and a 4-([4-(dimethylamino)phenyl]
azo)benzoic acid (DABCYL) quencher, this SDR catalytic system exhibits a
switch-on" response for ATP. Conditions for detecting ATP, such as the loading of
the "catalyst", buffer concentration of Mg2+ and incubation temperature, have been
optimized to afford a detection limit of 50 nM and a linear response up to 1400 nM of
ATP. This target inhibited catalytic cycle provides an enzyme-free biosensing strategy with higher sensitivity than many aptamer-based biosensing systems and even some
enzyme-based amplification systems.
5. The problems, which exist in the above target inhibited catalytic cycle-based
sensing system, are the multi-step operations and time-consuming detection process
(8 h). Therefore, a target triggered catalytic cycle was designed for biosensing.
Besides the SDR-based catalytic cycle, this new system also contains a target-induced
strand displacement process which releases the catalyst strand for the cycle from the
ATP aptamer-"Catalyst-2" duplex. The sensing of ATP was achieved by labeling
Substrate-2" and "C4" strands with FAM and DABCYL respectively. The addition of
ATP triggered the release of "Catalyst-2" so as to catalyze the SDR-based catalytic
cycle. Then, the bounded "Substrate-2" and "C4" strands in "S-C-2" complex were
separated in the presence of ATP and resuming the fluorescence. The intermediates in
the system were analyzed by PAGE and the time course of the catalysis process was
followed in the FAM and DABCYL labeled system. After optimizing the Mg2+
concentration, this target triggered catalytic-based sensing system provided a more
sensitive response (Limit of detection is 20 nM) to ATP than the target inhibited one.
It also provides a faster detection process (less than 1 hour's detection process) which
overcomes the disadvantage in the target inhibited catalytic cycle-based system.
| Date of Award | 14 Feb 2014 |
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| Original language | English |
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| Awarding Institution | - City University of Hong Kong
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| Supervisor | Hon Wah Michael LAM (Supervisor) |
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