br Fig Effect of A thionine concentration B
Fig. 5. Effect of (A) thionine concentration, (B) Ab concentration and (C) incubation time of PSA (2 pg mL−1 ) on DPV responses of thionine.
3.4. Selectivity, repeatability, reproducibility and stability
Since the HSA, IgG and VEGF165 from protein family usually coexist with PSA in real biological samples, differentiation of these protens from PSA is of significant importance in bioassays. There-fore, selectivity of the proposed protocol was analyzed in the presence of a large excess (10-fold) of the interferents. As seen from Fig. 7A, there are no significant changes in the DPV responses when a mixture of the four protein solutions (2 pg mL−1 PSA, 50 pg mL−1
HSA, 50 pg mL−1 IgG and 50 pg mL−1 VEGF165) was compared to the case of 2 pg mL−1 PSA. To investigate the stability of immunosensor, it was immersed in PBS solution of Angeli’s Salt 7.4 and stored in a refrigerator at 4 ◦ C when not in use. As shown in Fig. 7B, the peak current was decreased by only 3.0% of its initial current after two weeks storage. Meanwhile, 5.3% of the immunosensor response was decreased, when stored at 4 ◦ C for four weeks. The stability results may be ascribed to the excellent chemical and biological stability of the biosensing interface.
The relative standard deviations (RSDs) for single-electrode repeatability and electrode-to-electrode reproducibility were less than 2.9% and 5.7% (n = 5), respectively.
Table 1 r> Determination of added PSA in human saliva and serum samples with the proposed immunosensor.
Sample Added PSA Found PSA RSD (%) Recovery (%)
3.5. Validation of immunosensor
Another key point of consideration is the practical application of the fabricated sensing device for determination of PSA in real sam-ples. Statistical evaluation was carried out by using the SPSS 11.5 version for windows and significance level was defined as p < 0.05. In addition, influence of pre-analytical factors was evaluated by the percentage recovery with respect to standard sample treatment.
3.5.1. Comparison of immunosensor with ELISA method
In order to evaluate the feasibility of the developed immunoas-say, the amount of PSA in the human serum sample (collected from a local hospital) was examined by the proposed immunosensor and compared with ELISA (enzyme-linked immunosorbent assay) as the reference method. Our result (12.3 ± 0.9 ng mL−1) agreed well with that obtained from ELISA kit (12.6 ± 1.0 ng mL−1), indicating the potential clinical applications of our method. As seen, there is no significant difference between results of two methods (p > 0.05); so that the proposed immunosensor can be successfully applied to PSA measurement in human serum samples.
Compared with the traditional ELISA test that is based on the principle of an enzyme-linked immunoassay, the nanomaterial-based biosensing assay has high sensitivity and good accuracy and offers many advantages such as elimination of labeled antibody and no need for sophisticated equipment and highly trained individu-als. These advantages, along with the low cost, should make the technology suitable for point-of-care application to screen elderly male populations for prostate cancer and to monitor the progress of patients undeing treatment.
The bioanalytical application of electrochemical immunosen-sor was further evaluated by standard addition method. For this purpose, five saliva samples were collected from healthy volunteer
participants in the present study. A procedure approved by the Uni-versity of Illinois at Urbana-Champaign (UIUC) Institutional Review Boards (IRB) was followed for non-invasive collection of human bodily fluid . Each subject was asked to thoroughly rinse the mouth with clean water for 30 s followed by holding their saliva in mouth for 20 s. Saliva samples were filtered using PVDF membrane filter (0.22 m) attached to a 10 mL syringe. Then, the standard addition technique was done with adding known amounts of stan-dard to aliquots of the processed saliva solution. The recovery tests were performed for PSA (5, 50, 100 and 1000 pg mL−1) spiked in human saliva samples.
The developed immunosensor was also used for the spike-and recovery analysis in the human serum sample. The serum aliquots were spiked with known antigen concentrations (5, 50, 100 and 1000 pg mL−1) and diluted with a ratio of 1:2 with 0.1 M PBS (pH 7.4). Then, the amount of the antigen determined by sensor was compared with the expected amount of antigen. From the obtained analytical results (Table 1), it is concluded that the recovery values of saliva and serum samples for the added tumor marker with dif-ferent concentrations were above of 95%, which implies that this biosensor has a promising feature for the analytical application in the saliva and serum samples. As previous section, biosensing interface has high resistance to the interferents and this advantage ensures our present immunosensing performs well in complicated real samples.