Reagentless, reusable, ultrasensitive electrochemical molecular beacon aptasensor.
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.
Radi
Abd-Elgawad
AE
Nanobiotechnology and Bioanalysis Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Tarragona, Spain. abd.radi@urv.net
Acero Sánchez
Josep Lluis
JL
Baldrich
Eva
E
O'Sullivan
Ciara K
CK
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D015374:Biosensing Techniques; D017097:Electric Impedance; D004563:Electrochemistry; D004566:Electrodes; D005296:Ferrous Compounds / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D006531:HEPES / Q000737:chemistry; D000075163:Metallocenes; D009690:Nucleic Acid Conformation; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry
D052157:Aptamers, Nucleotide; D005296:Ferrous Compounds; D000075163:Metallocenes; D013438:Sulfhydryl Compounds; C081676:thrombin aptamer; D006046:Gold; D013917:Thrombin; D006531:HEPES; C004998:ferrocene