Potassium ferricyanide K₃[Fe(CN)₆], potassium ferrocyanide K₄[Fe(CN)₆], potassium dihydrogen phosphate, sodium monophosphate and the target protein thrombin (Thr), were purchased from Sigma (St. Louis, MO, USA). Poly(ethylene glycol) (PEG), sodium chloride and potassium chloride were purchased from Fluka (Buchs, Switzerland). All reagents were analytical reagent grade. All-solid-state electrodes (GECs) were prepared using 50 μm particle size graphite powder (Merck, Darmstadt, Germany) and Epotek H77 resin and its corresponding hardener (both from Epoxy Technology, Billerica, MA, USA). The aptamer (AptThr) used in this study, with sequence 5′-GGTTGGTGTGGTTGG-3′, was prepared by TIB-MOLBIOL (Berlin, Germany). All solutions were made up using MilliQ water from MilliQ System (Millipore, Billerica, MA, USA). The buffer employed was PBS (187 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄·2H₂O, 1.76 mM KH₂PO₄, pH 7.0). Stock solutions of aptamer and thrombin were diluted with sterilized and deionised water, separated in fractions and stored at −20 °C until used.

AC impedance measurements were performed with an IM6e Impedance Measurement Unit (BAS-Zahner, Kronach, Germany) and Autolab PGStat 20 (Metrohm Autolab B.V, Utrecht, The Netherlands). Thales (BAS-Zahner) and Fra (Metrohm Autolab) software, respectively, were used for data acquisition and control of the experiments. A three electrode configuration was used to perform the impedance measurements: a platinum-ring auxiliary electrode (Crison 52–67 1, Barcelona, Spain), an Ag/AgCl reference electrode and the constructed GEC as the working electrode. Temperature-controlled incubations were done using an Eppendorf Thermomixer 5436.

Graphite epoxy composite (GEC) electrodes used were prepared using a PVC tube body (6 mm i.d.) and a small copper disk soldered at the end of an electrical connector, as shown on Figure 1(a). The working surface is an epoxy-graphite conductive composite, formed by a mixture of graphite (20%) and epoxy resin (80%), deposited on the cavity of the plastic body [15,16]. The composite material was cured at 80 °C for 3 days. Before each use, the electrode surface was moistened with MilliQ water and then thoroughly smoothed with abrasive sandpaper and finally with alumina paper (polishing strips 301044-001, Orion) in order to obtain a reproducible electrochemical surface.

The analytical procedure for biosensing consists of the immobilization of the aptamer onto the transducer surface using a wet physical adsorption procedure, followed by the recognition of the thrombin protein by the aptamer via incubation at room temperature. The scheme of the experimental procedure is represented in Figure 1(b), with the steps described in more detail below.

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, −Z_im vs. Z_re) and fitted to a theoretical curve corresponding to the equivalent circuit with Z_view 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 R_ct) 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 (R_ct) 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 Δ_ratio value was defined according to the following equations: Δ ratio = Δ s / Δ p Δ s = R ct   ( AptThr-Thr ) − R ct   ( electrode-buffer ) Δ p = R ct   ( AptThr ) − R ct   ( electrode-buffer )where R_{ct(AptThr-Thr)} was the electron transfer resistance value measured after incubation with the thrombin protein; R_ct(AptThr) was the electron transfer resistance value measured after aptamer inmobilitation on the electrode, and R_{ct(electrode-buffer)} was the electron transfer resistance of the blank electrode and buffer.

First of all, the concentration of aptamer and PEG immobilized onto the electrode surface were optimized separately by building its response curves. For this, increasing concentrations of AptThr and PEG were used to carry out the immobilization, evaluating the changes in the Δ_p.

Figure 3 shows the curve of AptThr adsorption onto the electrode surface. It can be observed that the difference in resistance (Δ_p) 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 R_ct increases to reach a saturation value, chosen as the optimal concentration. This value corresponded to a concentration of aptamer of 1 μM.

To minimize any possible nonspecific adsorption onto the electrode surface, polyethylene glycol (PEG) was used as the blocking agent. As shown in Figure 4, and like the previous case, there was an increase in resistance until the value of 40 mM of PEG where the saturation value is reached. Therefore, the optimal concentration of blocking agent was chosen as 40 mM.

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 R_ct 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.

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 Δ_ratio vs. 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 (Δ_ratio) and the thrombin concentration in this range was obtained, according to the equation: Δ_ratio = 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].

Thrombin is present in blood serum, a complex sample matrix, with hormones, lipids, blood cells and other proteins [23]. To study the selectivity of the system, we evaluated the response of proteins typically present in serum such as fibrinogen, immunoglobulin G and albumin. In the first case, we tested albumin protein, which is found in serum at a level from 3,500 to 5,000 mg/dL [24], representing more than 60% of the total protein present. To perform the test, the highest concentration in serum was used, that is 5,000 mg /dL. When the aptamer was incubated with this protein, electron interfacial resistance did not increase, in this case it was observed a slight decrease. Afterwards, when the aptamer was incubated with thrombin, an increase in the resistance, and of expected magnitude, was observed. Therefore, it was proved that albumin was not recognized by the AptThr, and it did not interfere with aptamer-thrombin system.

In the second case, fibrinogen was evaluated as an interfering protein. Fibrinogen is a fibrillar protein involved in the blood clotting process. By the action of thrombin, fibrin is degraded and results in the formation of a clot [25]. This protein is present in human serum in a concentration range of 200 to 400 mg/dL [26]. It was observed that the electron interfacial resistance increased as a result of some type of recognition by the AptThr. Therefore, this protein could act as an interference for the system.

In the last case, generic immunoglobulin G (IgG) was used. IgG is a globular protein that is synthesized in response to the invasion of any bacteria, virus or fungi. It is present in human serum over a range of concentrations from 950 mg/dL to 1,550 mg/dL in serum, with a normal value of 1,250 mg/dL. IgG also acted as interferent, which is proved from the increase of the resistance R_ct. This increase, as it also happened in the case of fibrinogen, may be due to some biological interaction between the aptamer and these proteins, not yet described. In the last two cases, the addition of thrombin to the system increased the interfacial resistance between the electrode and the surface; this fact could be due to some phenomenon of partial displacement between thrombin and protein interferents that could take place.

To evaluate the sensitivity of the aptasensor we compared the calibration plots for the different proteins. Table 1 summarizes the parameters of the calibration curve of each protein and thrombin, as well as their respectives slopes and detection limits. The aptasensor showed the highest sensitivity for its target molecule, thrombin, with its slope being 6 orders of magnitude greater than the slope for IgG and five orders of magnitude more than the one for fibrinogen, as shown in Figure 8. Therefore, it was demostrated that the aptasensor showed a much higher sensitivity to Thr, regarding potential interfering proteins, which displayed this effect due to the high level of concentration in which they are present.

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 R_ct 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 Δ_ratio on every step of the process and represented in the bar chart as shown in Figure 9. In the third incubation with Thr, Δ_ratio 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.