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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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Calibration curve vs. thrombin concentration. (Δratio = Δs /Δp; Δs = Rct(AptThr-Thr) − Rct (electrode-buffer); Δp = Rct (AptThr) − Rct(electrode-buffer)).
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f7-sensors-12-03037: Calibration curve vs. thrombin concentration. (Δratio = Δs /Δp; Δs = Rct(AptThr-Thr) − Rct (electrode-buffer); Δp = Rct (AptThr) − Rct(electrode-buffer)).

Mentions: Performing new experiments with solutions containing different amount of thrombin, the calibration curve was built. Figure 6 shows the evolution of the Nyquist diagrams for the calibration of the aptasensor. There is a correct recognition of the protein by the aptamer; as by increasing thrombin concentration, the interfacial electron transfer resistance between the electrode surface and solution also increases, until reaching saturation. To evaluate the linear range and detection limit of the AptThr-Thr system, the calibration curve was built, representing the analytical signal expressed as Δratiovs. the protein concentration (Figure 7). As can be seen, a sigmoidal trend is obtained, where the central area could be approximated to a straight line, with a linear range from 7.5 pM to 75 pM for the protein. Moreover, a good linear relationship (r2 = 0.9981) between the analytical signal (Δratio) and the thrombin concentration in this range was obtained, according to the equation: Δratio = 1.013 + 1.106 × 1010 [Thr]. The EC50 was estimated as 44 pM and the detection limit, calculated as three times the standard deviation of the intercept obtained from the linear regression, was 4.5 pM. The reproducibility of the method showed a relative standard deviation (RSD) of 7.2%, obtained from a series of 5 experiments carried out in a concentration of 75 pM Thr. These are satisfactory results for the detection of thrombin in real samples, given this level is exactly the concentration threshold when forming thrombus [9].


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

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

Calibration curve vs. thrombin concentration. (Δratio = Δs /Δp; Δs = Rct(AptThr-Thr) − Rct (electrode-buffer); Δp = Rct (AptThr) − Rct(electrode-buffer)).
© Copyright Policy
Related In: Results  -  Collection

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

f7-sensors-12-03037: Calibration curve vs. thrombin concentration. (Δratio = Δs /Δp; Δs = Rct(AptThr-Thr) − Rct (electrode-buffer); Δp = Rct (AptThr) − Rct(electrode-buffer)).
Mentions: Performing new experiments with solutions containing different amount of thrombin, the calibration curve was built. Figure 6 shows the evolution of the Nyquist diagrams for the calibration of the aptasensor. There is a correct recognition of the protein by the aptamer; as by increasing thrombin concentration, the interfacial electron transfer resistance between the electrode surface and solution also increases, until reaching saturation. To evaluate the linear range and detection limit of the AptThr-Thr system, the calibration curve was built, representing the analytical signal expressed as Δratiovs. the protein concentration (Figure 7). As can be seen, a sigmoidal trend is obtained, where the central area could be approximated to a straight line, with a linear range from 7.5 pM to 75 pM for the protein. Moreover, a good linear relationship (r2 = 0.9981) between the analytical signal (Δratio) and the thrombin concentration in this range was obtained, according to the equation: Δratio = 1.013 + 1.106 × 1010 [Thr]. The EC50 was estimated as 44 pM and the detection limit, calculated as three times the standard deviation of the intercept obtained from the linear regression, was 4.5 pM. The reproducibility of the method showed a relative standard deviation (RSD) of 7.2%, obtained from a series of 5 experiments carried out in a concentration of 75 pM Thr. These are satisfactory results for the detection of thrombin in real samples, given this level is exactly the concentration threshold when forming thrombus [9].

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