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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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Equivalent circuit used for the data fitting. R1 is the resistance of the solution, R2 is the electron-transfer resistance and CPE, the capacitive contribution, in this case as a constant phase element.
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f2-sensors-12-03037: Equivalent circuit used for the data fitting. R1 is the resistance of the solution, R2 is the electron-transfer resistance and CPE, the capacitive contribution, in this case as a constant phase element.

Mentions: Impedimetric measurements were performed in 0.01 mM [Fe(CN)6]3−/4− solution prepared in PBS at pH 7. The electrodes were dipped in this solution and a potential of +0.17 V (vs. Ag/AgCl) was applied. Frequency was scanned from 10 kHz to 50 mHz with a fixed AC amplitude of 10 mV. The impedance spectra were plotted in the form of complex plane diagrams (Nyquist plots, −Zimvs. Zre) and fitted to a theoretical curve corresponding to the equivalent circuit with Zview software (Scribner Associates Inc., USA). In the equivalent circuit shown in Figure 2, the parameter R1 corresponds to the resistance of the solution, R2 is the charge transfer resistance (also called Rct) between the solution and the electrode surface, whilst CPE is associated with the double-layer capacitance (due to the interface between the electrode surface and the solution). The use of a constant phase element (CPE) instead of a capacitor is required to optimize the fit to the experimental data, and this is due to the nonideal nature of the electrode surface [14]. The parameters of interest in our case are the electron-transfer resistance (Rct) and the chi-square (χ2). The first one was used to monitor the electrode surface changes, while χ2 was used to measure the goodness of fit of the model. In all cases the calculated values for each circuit were <0.2, much lower than the tabulated value for 50 degrees of freedom (67.505 at 95% confidence level). In order to compare the results obtained from the different electrodes used, and to obtain independent and reproducible results, relative signals are needed [16]. Thus, the Δratio value was defined according to the following equations:Δratio=Δs/ΔpΔs=Rct (AptThr-Thr)−Rct (electrode-buffer)Δp=Rct (AptThr)−Rct (electrode-buffer)where Rct(AptThr-Thr) was the electron transfer resistance value measured after incubation with the thrombin protein; Rct(AptThr) was the electron transfer resistance value measured after aptamer inmobilitation on the electrode, and Rct(electrode-buffer) was the electron transfer resistance of the blank electrode and buffer.


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

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

Equivalent circuit used for the data fitting. R1 is the resistance of the solution, R2 is the electron-transfer resistance and CPE, the capacitive contribution, in this case as a constant phase element.
© Copyright Policy
Related In: Results  -  Collection

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

f2-sensors-12-03037: Equivalent circuit used for the data fitting. R1 is the resistance of the solution, R2 is the electron-transfer resistance and CPE, the capacitive contribution, in this case as a constant phase element.
Mentions: Impedimetric measurements were performed in 0.01 mM [Fe(CN)6]3−/4− solution prepared in PBS at pH 7. The electrodes were dipped in this solution and a potential of +0.17 V (vs. Ag/AgCl) was applied. Frequency was scanned from 10 kHz to 50 mHz with a fixed AC amplitude of 10 mV. The impedance spectra were plotted in the form of complex plane diagrams (Nyquist plots, −Zimvs. Zre) and fitted to a theoretical curve corresponding to the equivalent circuit with Zview software (Scribner Associates Inc., USA). In the equivalent circuit shown in Figure 2, the parameter R1 corresponds to the resistance of the solution, R2 is the charge transfer resistance (also called Rct) between the solution and the electrode surface, whilst CPE is associated with the double-layer capacitance (due to the interface between the electrode surface and the solution). The use of a constant phase element (CPE) instead of a capacitor is required to optimize the fit to the experimental data, and this is due to the nonideal nature of the electrode surface [14]. The parameters of interest in our case are the electron-transfer resistance (Rct) and the chi-square (χ2). The first one was used to monitor the electrode surface changes, while χ2 was used to measure the goodness of fit of the model. In all cases the calculated values for each circuit were <0.2, much lower than the tabulated value for 50 degrees of freedom (67.505 at 95% confidence level). In order to compare the results obtained from the different electrodes used, and to obtain independent and reproducible results, relative signals are needed [16]. Thus, the Δratio value was defined according to the following equations:Δratio=Δs/ΔpΔs=Rct (AptThr-Thr)−Rct (electrode-buffer)Δp=Rct (AptThr)−Rct (electrode-buffer)where Rct(AptThr-Thr) was the electron transfer resistance value measured after incubation with the thrombin protein; Rct(AptThr) was the electron transfer resistance value measured after aptamer inmobilitation on the electrode, and Rct(electrode-buffer) was the electron transfer resistance of the blank electrode and buffer.

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