Fluxametamide br In order to prove the
In order to prove the superior performance of the MWCNT on the electrode surface, the CV of thionine immobilized on the GCE/MWCNT/His-rGO was compared with that on the GCE/His-rGO. As seen, the MWCNT/His-rGO/thionine biointerface possesses a larger CV current than the His-rGO/thionine biointerface, which attributed to the fact that the CNTs facilitated the electron transfer between redox probe and the GCE . In addition the stability of bifunctional rGO on the electrode surface was efficiently improved by employing CNTs.
To reconfirm the stepwise assembly of the immunosensor from the view of impedance changes of the electrode surface, EIS studies were performed in 5.0 mM [Fe(CN)6]3−/4− containing 0.1 M KCl. The semicircle diameter at higher frequencies represents the electrode transfer resistance (Ret) and the linear portion at low frequen-cies represents the Fluxametamide step of the overall process . As
Fig. 2. Schematic illustration of the stepwise immunosensor fabrication process.
shown in Fig. 4, the bare GCE exhibited a small Ret value (curve
a) corresponding to low semicircle diameter because of its cer-tain conductivity. After that dropping of carbon nanotubes on the surface of the GCE (curve b), the Ret value was smaller than the bare GCE, owing to the excellent conductivity of MWCNTs . Then, a decrease Ret was observed when the GCE/MWCNT was further modified with His-rGO (curve c), suggesting that the func-tionalized graphene is successfully immobilized on the electrode surface and the bifunctional rGO film can enhance the electron transfer. When thionine was assembled on the electrode surface, Ret of GCE/MWCNT/His-rGO/thionine (curve d) was significantly decreased compared with that of the GCE/MWCNT/His-rGO, which implied that thionine acted as the electron transfer mediator . It was also observed that the resistance increased gradually with sequential immobilization of antibody (curve e) and PSA antigen (curve f). The gradual increased Ret with the addition of Ab and PSA revealed the fact that the proteins can hinder the electronic con-
ductivity. In fact, the increase in resistance is due to the formation of protein layers on the surface of the electrode, which impeded the electron transfer of [Fe(CN)6]3−/4− on the GCE surface .
3.2. Optimization of immunosensing parameters
In this study, the effect of experimental parameters such as the concentration of thionine, Ab density and target incubation time on the immunosensor response was investigated.
The concentration of redox indicator plays an important role in the sensing performance. As shown in Fig. 5A, the current response boosted drastically with an increase in thionine concentration up to 1 mM and then reached a plateau. So, an optimum probe con-centration of 1 mM was used for further studies.
Since the bioreceptor density can be controlled by varying the concentration of antibody used in the sensor fabrication; thus, the influence of concentration on the response characteristics of the
Fig. 3. (A) Electrochemical characterization of the PSA immunosensor by cyclic voltammetry in 0.1 M PBS of pH 7.4 and (B) electrochemical reduction of thionine on the GCE/MWCNT/His-rGO in the absence or presence of PSA specific antibody.
prepared sensor was studied and the results are shown in Fig. 5B. As is obvious, the current response decreased with the increasing concentration from 0.5 M to 1.0 M, while further increasing con-centration did not significantly change the signal. Therefore, 1 M was selected as the optimal Ab concentration.
The time of target incubation is a vital influencing factor on the response of the biosensors. As is seen in Fig. 5C, the current response decreased rapidly with increasing incubation time up to 25 min and leveled off at higher time periods, suggesting that the formation of antigen-antibody complex has reached to a saturation level on the electrode surface.
3.3. Voltammetric detection of PSA
Under the optimum conditions, the GCE/His-rGO/thionine/Ab was introduced to various concentrations of PSA. After incubation with the target, the DPV voltammograms of immunosensor were recorded in 0.1 M PBS pH 7.4. Consequently, the peak current value decreased with increasing concentration of tumor marker, which clearly shows a “signal-off” trend (Fig. 6). Insert of Fig. 6 shows a good linear relationship between the DPV peak currents of thion-ine and logarithmic concentration of PSA from 10 fg mL−1 to 20 ng mL−1. The linear regression equation was Ip( A) = −5.75 log ([PSA] (pg mL−1)) + 31.82, with a correlation coefficient of 0.996. The limit of detection (LOD) and limit of quantification (LOQ) for analytical procedure evaluated as 3Sb/m and 10Sb/m were 2.8 fg mL−1 and 9.3 fg mL−1, respectively, where Sb is the relative standard devia-tion of peak current corresponding to blank sample (n = 5) and m is the slope of the calibration plot. These results indicated that the proposed immunosensor can successfully detect the PSA antigen with a wide linear range and a very low LOD.