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A reusable impedimetric aptasensor for detection of thrombin employing a graphite-epoxy composite electrode.

Ocaña C, Pacios M, del Valle M - Sensors (Basel) (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain. cristina.ocana@uab.es

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.

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Nyquist Diagram of: (a) Electrode-buffer •, (b) Aptamer of thrombin (AptThr) , and (c) AptThr-Thr ○ 10 pM [Thr]. The arrow in each spectrum denotes the frequency (AC) of 10.9 Hz.
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f5-sensors-12-03037: Nyquist Diagram of: (a) Electrode-buffer •, (b) Aptamer of thrombin (AptThr) , and (c) AptThr-Thr ○ 10 pM [Thr]. The arrow in each spectrum denotes the frequency (AC) of 10.9 Hz.

Mentions: With the optimized concentrations of aptamer and PEG and following the above experimental protocol for the detection of thrombin, the aptasensor response was initially evaluated. The aptamer of thrombin forms a single-strand oligonucleotide, a chain that recognizes the protein by a three-dimensional folding (quadriplex). During this folding, weak interactions between the aptamer and protein of the host-guest type are created, leading to complex AptThr-Thr [22]. One example of the obtained response after each biosensing step is shown in Figure 5. As can be seen, resistance Rct between the electrode surface and the solution is increased. This fact is due to the effect on the kinetics of the electron transfer redox marker [Fe(CN)6]3−/[Fe(CN)6]4− which is delayed at the interface of the electrode, mainly caused by steric hindrance and electrostatic repulsion presented by the complex formed.


A reusable impedimetric aptasensor for detection of thrombin employing a graphite-epoxy composite electrode.

Ocaña C, Pacios M, del Valle M - Sensors (Basel) (2012)

Nyquist Diagram of: (a) Electrode-buffer •, (b) Aptamer of thrombin (AptThr) , and (c) AptThr-Thr ○ 10 pM [Thr]. The arrow in each spectrum denotes the frequency (AC) of 10.9 Hz.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376577&req=5

f5-sensors-12-03037: Nyquist Diagram of: (a) Electrode-buffer •, (b) Aptamer of thrombin (AptThr) , and (c) AptThr-Thr ○ 10 pM [Thr]. The arrow in each spectrum denotes the frequency (AC) of 10.9 Hz.
Mentions: With the optimized concentrations of aptamer and PEG and following the above experimental protocol for the detection of thrombin, the aptasensor response was initially evaluated. The aptamer of thrombin forms a single-strand oligonucleotide, a chain that recognizes the protein by a three-dimensional folding (quadriplex). During this folding, weak interactions between the aptamer and protein of the host-guest type are created, leading to complex AptThr-Thr [22]. One example of the obtained response after each biosensing step is shown in Figure 5. As can be seen, resistance Rct between the electrode surface and the solution is increased. This fact is due to the effect on the kinetics of the electron transfer redox marker [Fe(CN)6]3−/[Fe(CN)6]4− which is delayed at the interface of the electrode, mainly caused by steric hindrance and electrostatic repulsion presented by the complex formed.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain. cristina.ocana@uab.es

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.

Show MeSH
Related in: MedlinePlus