Abstract
Here, we report the application of a label-free electrochemical aptasensor based on a graphite-epoxy composite electrode for the detection of thrombin; in this work, aptamers were immobilized onto the electrodes surface using wet physical adsorption. The detection principle is based on the changes of the interfacial properties of the electrode; these were probed in the presence of the reversible redox couple [Fe(CN)6]3−/[Fe(CN)6]4− using impedance measurements. The electrode surface was partially blocked due to formation of aptamer-thrombin complex, resulting in an increase of the interfacial electron-transfer resistance detected by Electrochemical Impedance Spectroscopy (EIS). The aptasensor showed a linear response for thrombin in the range of 7.5 pM to 75 pM and a detection limit of 4.5 pM. The aptasensor was regenerated by breaking the complex formed between the aptamer and thrombin using 2.0 M NaCl solution at 42 °C, showing its operation for different cycles. The interference response caused by main proteins in serum has been characterized.
Highlights
Aptamers are artificial DNA or RNA oligonucleotides selected in vitro which have the ability to bind to proteins, small molecules or even whole cells, recognizing their target with affinities and specificities often matching or even exceeding those of antibodies [1]
The concentration of aptamer and polyethylene glycol (PEG) immobilized onto the electrode surface were optimized separately by building its response curves
It can be observed that the difference in resistance (Δp) increased up to a value. This is due to the physical adsorption of the aptamer onto the electrode surface, which followed a Langmuir isotherm; in it, the variation of Rct increases to reach a saturation value, chosen as the optimal concentration
Summary
Aptamers are artificial DNA or RNA oligonucleotides selected in vitro which have the ability to bind to proteins, small molecules or even whole cells, recognizing their target with affinities and specificities often matching or even exceeding those of antibodies [1]. The recognition process can be inverted and is stable in broad terms. Due to all these properties, aptamers can be used in a wide range of applications, such as therapeutics [2], molecular switches [3], drug development [4], affinity chromatography [5] and biosensors [6]. Concentration levels of thrombin in blood are very low, and levels down to picomolar range are associated with disease; because of this, it is important to be able to assess this protein concentration at trace level, with high selectivity [9]
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