16
L. Hao and H. Gu
and 15, respectively. One negative SELEX was also incorporated for the glutathione
magnetic beads at the beginning. Then, two aptamers N13 and N53 specific for
human FABP3 were isolated with corresponding K d of 0.0743 ± 0.0142 μM and
0.3337 ± 0.1485 μM for FABP3 interactions.
1.3.5 Introducing Evaluation into the Selection Process
1.3.5.1 SPR-SELEX
Although there have been verities of methods to select aptamer with various priorities, additional analytical methods were still required to evaluate the binding interaction between the ssDNA and the target during the process of SELEX [54]. The
introduction of real-time technologies to the process of SELEX can be an effective
alternative to monitor the events as they happen, which is more effective and efficient
for selecting aptamer with higher affinity [55].
Surface Plasmon Resonance (SPR) technique utilizes evanescent waves generated
to excite surface plasmons at the metal–dielectric (sensing medium) interface. SPR
is a conveniently real-time technique to evaluate the binding properties of DNA
pools during the SELEX process. Ngubane et al. [56] performed a surface plasmon
resonance-based SELEX to select RNA aptamers that bind to the ESX-3 secreted
protein, EsxG. EsxG was covalently immobilized on the chip using amine coupling
chemistry, and each concentration in the dilution series of the aptamer was injected
to detect the affinity. Two of the aptamers selected in this study, G43 and G78, were
analyzed for equilibrium kinetics and had Kd in the nanomolar range. Aptamer G43
had a Kd of 8.04 ± 1.90 nM while G78 had a Kd of 78.85 ± 9.40 nM.
However, early work was hardly to detect the signal in the first round of screening
[55, 57]. Dausse et al. [55] performed SPR-microfluidic SELEX for selection of
RNA aptamer of protein IRE1. During the dissociation phase of the SPR analysis,
aptamer candidates were recovered from the target immobilized onto the sensor chip
surface. The evaluation of the pools was simultaneously performed by SPR. An
obvious advantage of the method compared with methods in which the evaluation is
performed at the end of the selection is that since the pools are assayed for binding
in real time, after each round, the selection can be stopped as soon as the evolution
stops. In this SPR coupled SELEX method, the monitoring the affinity from the first
to the last round allowed facile identifying RNA aptamers that formed highly stable
loop–loop complexes (Kd equal to 8 nM) with the hairpin located on the 5
side of
the target.
Compared to Au, Ag nanoparticles generate sharper stronger plasmon resonance,
and Ag nanoparticles and alloy nanoparticles [58] are reported to generate larger
signal enhancements [59]. To further improve the selection efficiency of SPRSELEX, SPR imaging (SPRI), Ag10-NPs coupled DNA pool were integrated as
SPRI-SELEX for the selection of Lactoferrin (Lac) aptamer [60] (Fig. 1.11). Besides,
L. Hao and H. Gu
and 15, respectively. One negative SELEX was also incorporated for the glutathione
magnetic beads at the beginning. Then, two aptamers N13 and N53 specific for
human FABP3 were isolated with corresponding K d of 0.0743 ± 0.0142 μM and
0.3337 ± 0.1485 μM for FABP3 interactions.
1.3.5 Introducing Evaluation into the Selection Process
1.3.5.1 SPR-SELEX
Although there have been verities of methods to select aptamer with various priorities, additional analytical methods were still required to evaluate the binding interaction between the ssDNA and the target during the process of SELEX [54]. The
introduction of real-time technologies to the process of SELEX can be an effective
alternative to monitor the events as they happen, which is more effective and efficient
for selecting aptamer with higher affinity [55].
Surface Plasmon Resonance (SPR) technique utilizes evanescent waves generated
to excite surface plasmons at the metal–dielectric (sensing medium) interface. SPR
is a conveniently real-time technique to evaluate the binding properties of DNA
pools during the SELEX process. Ngubane et al. [56] performed a surface plasmon
resonance-based SELEX to select RNA aptamers that bind to the ESX-3 secreted
protein, EsxG. EsxG was covalently immobilized on the chip using amine coupling
chemistry, and each concentration in the dilution series of the aptamer was injected
to detect the affinity. Two of the aptamers selected in this study, G43 and G78, were
analyzed for equilibrium kinetics and had Kd in the nanomolar range. Aptamer G43
had a Kd of 8.04 ± 1.90 nM while G78 had a Kd of 78.85 ± 9.40 nM.
However, early work was hardly to detect the signal in the first round of screening
[55, 57]. Dausse et al. [55] performed SPR-microfluidic SELEX for selection of
RNA aptamer of protein IRE1. During the dissociation phase of the SPR analysis,
aptamer candidates were recovered from the target immobilized onto the sensor chip
surface. The evaluation of the pools was simultaneously performed by SPR. An
obvious advantage of the method compared with methods in which the evaluation is
performed at the end of the selection is that since the pools are assayed for binding
in real time, after each round, the selection can be stopped as soon as the evolution
stops. In this SPR coupled SELEX method, the monitoring the affinity from the first
to the last round allowed facile identifying RNA aptamers that formed highly stable
loop–loop complexes (Kd equal to 8 nM) with the hairpin located on the 5
side of
the target.
Compared to Au, Ag nanoparticles generate sharper stronger plasmon resonance,
and Ag nanoparticles and alloy nanoparticles [58] are reported to generate larger
signal enhancements [59]. To further improve the selection efficiency of SPRSELEX, SPR imaging (SPRI), Ag10-NPs coupled DNA pool were integrated as
SPRI-SELEX for the selection of Lactoferrin (Lac) aptamer [60] (Fig. 1.11). Besides,