本研究利用幾丁聚醣沉積層作為電化學式酵素免疫分析法中用以吸附捕捉抗體於白金電極上之材料，且成功製備出可增進電化學式雌二醇酵素免疫分析靈敏度之感測電極。在此檢測方法中，選定捕捉捕捉抗體為羊雌二醇抗體且以物理吸附的方式沉積在白金電極的幾丁聚醣沉積層上，偵測抗體以鼠雌二醇抗體及二次抗體為羊抗鼠免疫球蛋白交聯鹼性磷酯酶為組合的三明治式雌二醇酵素免疫分析法，二次抗體上酵素之基質與產物選定為4-氨基苯基磷酸與對氨基苯酚，本技術中的電極我們選用穩定性極高的白金電極，且使用小尺寸的微電極降低生產成本，此白金電極搭配選定之酵素基質與產物，我們決定了此電化學式酵素免疫分析的偵測電位(由酵素產物於白金電極表面之氧化電位所決定)為+0.14 V (vs. Ag/AgCl)，將電極置於攪拌的三羥甲基氨基甲烷緩衝液(pH 9.0)中，施加+0.14 V的定電位進行電流量測。此感測電極靈敏度(感測極限)為0.27 pg/mL且有寬闊的感測範圍從0.27 pg/mL至100 ng/mL，本研究特別對黃體酮及17α-乙炔基雌二醇之交叉反應進行評估分別得到0.033%及3.35%。本研究也成功地建立血清的校正曲線，且本研究之製備方法簡單、方便、迅速、制式化，為適合進行商品化之技術。

A convenient method for fabricating a sensitive 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 produced in goat was used as the capture antibody which was immobilized on the chitosan coated Pt wire microelectrode, anti-17β estradiol 6 antibody produced in mouse was used as the detection antibody, and goat anti-mouse IgG conjugated with alkaline phosphatase (AP) was used as the secondary antibody. 4-Aminophenyl phosphate (4-APP) was chosen as the AP substrate and the oxidation potential of the electroactive AP product, 4-amino phenyl (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 sensitivity (limit of detection) of this assay was 0.27 pg/mL with a wide detection range from 0.27 pg/mL up to 100 ng/mL. The assay specificity evaluated by testing the cross-reactivity of the assay against progesterone and 17α-ethynylestradiol was found to be 0.033% and 3.35%, respectively. The calibration curve of this assay tested in serum was also established. In this study, a sensitive, inexpensive, disposable, and easy to be fabricated electrochemical ELISA for estradiol was demonstrated.

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