Electronic ‘Off‐On’ Molecular Switch for Rapid Detection of Thrombin

Abstract

An electrochemical molecular beacon aptasensor for fast and sensitive assay of thrombin is developed and characterized. A bifunctional derivative of the thrombin-binding aptamer, 15-base long with a redox-active ferrocene moiety and a pendant hexanethiol linker group at the termini of the aptamer strand was immobilized on Au electrode surface. The electrochemical behavior of the aptasensor was studied using cyclic voltammetry (CV), chronoamperometry (CA), chronopotentiometry (CP), square-wave voltammetry (SWV) and chronopotentiometric stripping analysis (PSA) techniques. Taken together, these experiments support the switch "on" mechanism of the system when the thrombin binds to the immobilized aptamer. The variation of response to the concentration of thrombin, the target protein, was evaluated by square-wave voltammetry and chronopotentiometric stripping analysis in HEPES buffer solution (pH 8.0, 0.01 M). The aptasensor showed a specific response to thrombin in the range 1.0 to 35 nM with a detection limit of subnanomolar level concentrations. The reaction time, reproducibility, specificity and stability of the aptasensor were also studied.

Voltammetric aptasensors for protein disease biomarkers detection: A review

“…Upon binding with thrombin, a biotinylated secondary aptamer was added, which was ready to bind in a further step to a streptavidin-AP conjugate. A detection limit of 0.5 nM was obtained which is much lower than the values reported by the other authors previously mentioned that developed aptasensors based on target binding-induced aptamer conformational changes (Lai et al, 2007;Ma et al, 2013;Sánchez et al, 2006;Xiao et al, 2005a). These authors used the same strategy for detecting the CRP protein.…”

“…However, the aptasensors based in "signal-off" are usually less sensitive, more susceptible to background variation and small signal changes (Zhou et al, 2014). In the literature, the detection "signal-on" or "signal-off" is usually associated with target binding-induced aptamer conformational change Lai et al, 2007;Liu et al, 2010;Ma et al, 2013;Sánchez et al, 2006;Xiao et al, 2005a) and target binding-induced aptamer-complementary sequence dissociation/displacement strategies (Lu et al, 2008;Xiao et al, 2005b). This is due to the fact that aptamer-label or aptamer-complementary sequence-label after binding to its target promotes the approximation of the label on the electrode surface, thus increasing the current ("signal-on") or a distancing of the label from the Fig.…”

“…In the same work, a gold-plated screen-printed carbon electrode was tested showing a similar performance. Sánchez et al (2006) and Radi et al (2006) implemented the target binding-induced aptamer conformation principle to develop "signalon" electrochemical aptasensors to detect thrombin. The aptamers were modified with Fc and SWV or DPV were used for the electrochemical measurements, respectively.…”

Guanine Quadruplex Electrochemical Aptasensors

“…Similar G4 electrochemical aptasensors for thrombin were developed in parallel, using ferrocene labels [77][78][79]. In another design, a beacon aptamer-based biosensor for thrombin showed a linear signal between 0 and 50.8 nM of thrombin, with a 0.999 correlation factor and an 11 nM detection limit [80].…”

Redox behaviour of G-quadruplexes

“…the thrombin binding produced a decrease in the faradaic current of the methylene blue redox label, and the sensor was enough selective to detect thrombin directly in blood serum with a thrombin detection limit of 20 nM. Similar G-quadruplex aptasensors for thrombin were developed in parallel, using ferrocene labels [68][69][70]. The signal-on aptasensor achieved an increase in signal of ∼300% with a saturated target and a detection limit of 3 nM [71].…”