| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 218244 | Journal of Electroanalytical Chemistry | 2015 | 9 Pages |
•A sandwich type, indirect amperometric ELISA for estradiol has been demonstrated.•Electrodeposited chitosan has been used to immobilize capture antibody on Pt for enhancing the detection signal.•EIS analysis of the pre-coated ELISA electrodes has been provided.•The assay has low detection limit, wide detection range, and high specificity for estradiol in buffer samples.•The assay has been tested with estradiol in spiked serum samples.
A simple and inexpensive method for fabricating a sensitive and compact indirect sandwich type electrochemical ELISA for the detection of estradiol using a chitosan electrodeposited platinum (Pt) wire microelectrode was proposed. In this assay, anti-17β estradiol antibody produced in goat (goat anti-estradiol Ab) was used as the capture antibody which was immobilized on the chitosan coated Pt wire microelectrode, anti-17β estradiol antibody produced in mouse (mouse anti-estradiol Ab) was used as the detection antibody, and goat anti-mouse IgG (immunoglobulin G) conjugated with alkaline phosphatase (AP) was used as the secondary antibody. The effect of pre-coated layers (chitosan, the capture antibody, and the blocking reagent BSA) on the electron transfer resistance (Ret) at the surface of ELISA electrodes has been investigated and analyzed by the electrochemical impedance spectrum (EIS). 4-Aminophenyl phosphate (4-APP) was chosen as the AP substrate and the oxidation potential of the electroactive AP product, 4-aminophenol (4-AP), on the Pt electrode was determined to be + 0.14 V (vs. Ag/AgCl). The electrochemical ELISA was detected by constant potential amperometry at + 0.14 V in the Tris buffer (pH 9.0). The limit of detection of this assay was 2.7 × 10− 1 pg/mL with a wide detection range from 2.7 × 10− 1 pg/mL up to 1.0 × 105 pg/mL. The assay specificity evaluated by testing the cross-reactivity of the assay for progesterone and 17α-ethynylestradiol was found to be 0.033% and 3.4%, respectively. This assay has been tested with estradiol in spiked serum samples; however, further pretreatment of serum samples may be required to enhance precision and recovery.
