Gold nanoparticles (nAu) have demonstrated to be an useful tool for the development of electrochemical immunosensors. Among the various configurations used, those based on the incorporation of nAu to composite mixtures have several advantages. First, nAu can the incorporated into the composite matrix by simple physical inclusion, thus making the electrode fabrication easy, fast and cheap, and giving rise to bioelectrodes easily renewable by polishing. Furthermore, nAu act as isolated areas for biomolecules adsorption surronded by conductive composite material. This facilitates the preparation of immunosensors with an easy and strict control of the immunoreagents immobilization conditions.
In this work, a novel and sensitive electrochemical immunosensor for the determination of testosterone is reported. The approach combines the use of nAu and carbon nanotubes-Teflon composite electrodes with direct attachment of anti-testosterone to the electrode surface. A competitive immunoassay between testosterone and testosterone labeled with peroxidase was employed, the determination of testosterone being accomplished by amperometry upon addition of hydrogen peroxide in the presence of catechol.
The experimental variables involved in the preparation of the immunosensor and in immunoassay protocol were optimized. The composite electrode was prepared by adding 90 µL of colloidal gold to a 50:50 CNTs:Teflon mixture. Then, 10 µL aliquots from a 15 µg/mL anti-testosterone solution was incubated onto nAu/CNTs/Teflon for 30 min at 37ºC, with further blocking with BSA. The detection of testosterone was performed by depositing 10 µL of a 1 µg/mL testosterone-HRP solution also containing testosterone, on the immunosensor surface. After incubation for 30 min at 37ºC, the immunosensor was immersed into a
A sigmoidal calibration plot for testosterone with a linear range between 0.1 and 10 ng/mL ( r = 0.993) was obtained The EC50 value was 0.6 ng/mL and the Hill slope, -1.2 ± 0.2. A limit of detection of 0.10 ng/mL testosterone was also obtained. The RSD value for 0.5 ng / mL testosterone (n=10) was 3.5 %. Furthermore, inter-assay reproducibility was evaluated by measuring testosterone concentration with nine immunosensors prepared in different days, with triplicate measurements every day, with a RSD value of 4.6 %. The lifetime of the immunosensor was evaluated by performing daily three measurements of a blank solution (without testosterone) with the anti-testosterone/nAu/CNTs/Teflon electrode. The results obtained indicated that the mean initial responses remained inside the control limits (± 3s) for eight days with no need for applying any regeneration procedure.
A cross-reactivity study for various compounds closely related with testosterone was also performed. No significative interference was observed for epi-testosterone, estradiol and 17α-methyl testosterone.
The electrochemical immunoassay was validated by analyzing human serum samples spiked with testosterone. Calibration graphs from serum samples were constructed giving similar curves to that of testosterone standard solutions. Various spiked samples containing 2.5 µg/mL testosterone were analyzed by interpolation into the calibration equation. A mean value (n=6) of 2.6 ± 0.2 ng/mL testosterone, with recoveries ranging between 86 and 112 %, and a mean recovery value of 98 ± 7% were obtained. These results demonstrate fairly well the usefulness of the imunosensor for the determination of testosterone at the concentration level required in this type of samples.
