SPRi在核酸适配体开发中的应用
HORIBA Scientific
G. Lautnera, R. Gyurcsanyia, K. Mercierb, E. Ly-Morinb
(a) Budapest University of Technology and Economics, Department of Inorganic and Analytical Chemistry, Budapest, Hungary
Aptamers are in vitro-generated, short, single-stranded nucleic acids that selectively bind to target compounds such as small molecules or proteins1, 2. Their main advantage over monoclonal antibo-dies is their resistance to denaturation and degradation.Being very robust and easy to synthesize, they are becoming widely used for the development of aptamer- based biosensors in the diagnost-ics field3.
Key words:
Material and Method
DNA aptamers
Immobilization of the human IgE (hIgE) aptamer on the SPRi-Biochip™
The thiol modified IgE aptamer and reference DNA was immobilized on the planar gold SPRi-Biochip™ sensor through thiol-Au chemisorption.The aptamer solutions were prepared at 10 μM in 20 mM PBS pH 7.4.Four rep-licate spots were printed simultaneously for each aptamer using the SPRi-CFM (Figure 1) during a 15-minute back-and-forth cycling.
The SPRi-CFM uses flow deposition to immobilize 48 molecules in a single run (up to 144 spots per chip). Immobilizing with flow generates increased spot homogeneity and higher immobilization levels.
After the immobilization procedure, the SPRi-Biochip™ surface was blocked with a 0.1 mM MUTEG solution pre-pared in 10 mM PBS pH 7.4. While MUTEG has good resistance against non-specific adsorption, it is sterically short enough not to hinder the specific binding event.
The spotted biochip was inserted into the SPRi-PlexII™ instrument from HORIBA Scientific. The system is equ-ipped with a 200 μL sample loop,a continuous flow pump and an in-line degasser.
The flow rate was set to 50 μL/min, and the working temperature was fixed at 25°C.Different concentrations of hIgE prepared in a two-fold dilution in the running buffer (10 mM PBS + 1 mM MgCl2) were injected in order to determine the affinity between hIgE and its aptamer (see Table 1).
The SPRi-Biochip™ surface was regenerated using a 12 mM NaOH with 1.2 % EtOH solution after each hIgE injection.
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Kinetics analysis of the aptamer-hIgE interaction
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This application note demonstrates that the SPR imaging platform from HORIBA Scientific is well adapted to the characterization of aptamerprotein interactions. The SPRi difference image gives meaningful information rega-rding the presence of binding and its specificity.
The realtime monitoring of the interaction gives access to the kinetics (associationand dissociation rates) and the affinity of the interaction. The multiplexing capabilities of the platform will bring very promising applications for the development of aptamer-based biochips in biomedical diag-
nostics.