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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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Signals obtained for three consecutive processes of regeneration. [Thr] used: 7.5 pM (1 and 2), and 75 pM (3). Uncertainty values corresponding to replicated experiments (n = 3).
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f9-sensors-12-03037: Signals obtained for three consecutive processes of regeneration. [Thr] used: 7.5 pM (1 and 2), and 75 pM (3). Uncertainty values corresponding to replicated experiments (n = 3).

Mentions: Finally, it was possible to regenerate the aptasensor by dissociating the AptThr-Thr complex, formed by weak interactions. It was achieved by stirring the aptasensor in saline media and increasing the temperature (42 °C). In this way, to show regeneration, three sensing cycles were performed with a blank measure in between each. Thrombin was added to the media and an increase of Rct due to complex formation AptThr-Thr was recorded. Then, by adding a saline buffer, increasing the temperature and stirring, the complex dissociated and resistance decreased to the baseline value of the correspondent (AptThr), and so on. Values were calculated as Δratio on every step of the process and represented in the bar chart as shown in Figure 9. In the third incubation with Thr, Δratio was increased more than in the other incubations, this was because it was incubated with a higher concentration. This type of regeneration may be an alternative to the polishing surface renewal, a typical feature of graphite-epoxy composite electrodes; an important advantage is that it regenerates the electrode surface without removing the immobilized aptamer on the electrode surface, which means that their use is largely facilitated and that cost of each analysis is reduced drastically.


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

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

Signals obtained for three consecutive processes of regeneration. [Thr] used: 7.5 pM (1 and 2), and 75 pM (3). Uncertainty values corresponding to replicated experiments (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

f9-sensors-12-03037: Signals obtained for three consecutive processes of regeneration. [Thr] used: 7.5 pM (1 and 2), and 75 pM (3). Uncertainty values corresponding to replicated experiments (n = 3).
Mentions: Finally, it was possible to regenerate the aptasensor by dissociating the AptThr-Thr complex, formed by weak interactions. It was achieved by stirring the aptasensor in saline media and increasing the temperature (42 °C). In this way, to show regeneration, three sensing cycles were performed with a blank measure in between each. Thrombin was added to the media and an increase of Rct due to complex formation AptThr-Thr was recorded. Then, by adding a saline buffer, increasing the temperature and stirring, the complex dissociated and resistance decreased to the baseline value of the correspondent (AptThr), and so on. Values were calculated as Δratio on every step of the process and represented in the bar chart as shown in Figure 9. In the third incubation with Thr, Δratio was increased more than in the other incubations, this was because it was incubated with a higher concentration. This type of regeneration may be an alternative to the polishing surface renewal, a typical feature of graphite-epoxy composite electrodes; an important advantage is that it regenerates the electrode surface without removing the immobilized aptamer on the electrode surface, which means that their use is largely facilitated and that cost of each analysis is reduced drastically.

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