A bifunctional derivative of the thrombin-binding aptamer with a redox-active Fc moiety and a thiol group at the termini of the aptamer strand was synthesized. The ferrocene-labeled aptamer thiol was self-assembled through S-Au bonding on a polycrystalline gold electrode surface and the surface was blocked with 2-mercaptoethanol to form a mixed monolayer. By use of a fluorescent molecular beacon, the effect of counterions on quadruplex formation was established. The aptamer-modified electrode was characterized electrochemically by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The modified electrode showed a voltammetric signal due to a one-step redox reaction of the surface-confined ferrocenyl moiety of the aptamer immobilized on the electrode surface in 10 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer of pH 8.0. An increase in the DPV current signal was evident after blocking with 2-mercaptoethanol, effectively removing aptamer nonspecifically absorbed rather than bound to electrode surface or due to the formation of the aptamer-thrombin affinity interaction. The impedance measurement, in agreement with the differential pulse voltammetry (DPV), showed decreased Faradaic resistances in the same sequence. The "signal-on" upon thrombin association could be attributed to a change in conformation from random coil-like configuration on the probe-modified film to the quadruplex structure. The DPV of the modified electrode showed a linear response of the Fc oxidation signal to the increase in the thrombin concentration in the range between 5.0 and 35.0 nM with a linear correlation of r = 0.9988 and a detection limit of 0.5 nM. The molecular beacon aptasensor was amenable to full regeneration by simply unfolding the aptamer in 1.0 M HCl, and could be regenerated 25 times with no loss in electrochemical signal upon subsequent thrombin binding.

A novel impedimetric aptasensor using a mixed self-assembled monolayer composed of thiol-modified thrombin binding aptamer and 2-mercaptoethanol on a gold electrode is reported. The changes of interfacial features of the electrode were probed in the presence of the reversible redox couple, Fe(CN)6(3-/4-), using impedance measurements. The electrode surface was partially blocked due to the self-assembly of aptamer or the formation of the aptamer-thrombin complex, resulting in an increase of the interfacial electron-transfer resistance detected by electrochemical impedance spectroscopy or cyclic voltammetry. The aptasensor was regenerated by breaking the complex formed between the aptamer and thrombin using 2.0 M NaCl solution, and the immobilized aptamer subsequently was used for repeated detection of thrombin. The aptamer-functionalized electrode showed a linear response of the charge-transfer resistance to the increase of thrombin concentration in the range of 5.0-35.0 nM and the thrombin was easily detectable to a concentration of 2.0 nM.

No abstract