A Miniaturized Impedimetric Immunosensor for the Competitive Detection of Adrenocorticotropic Hormone

Adrenocorticotropic hormone (ACTH) plays an essential role in regulating corticosteroid hormone production, which has important functions in a myriad of critical physiological functions. In this proof-of-concept study, a miniaturized immunosensor was developed for the highly sensitive detection of ACTH using electrochemical impedance spectroscopy (EIS) in connection with disposable screen-printed gold electrodes (SPGEs). A film of 3,3'-dithiobis[sulfosuccinimidylpropionate] (DTSSP) was prepared to immobilize anti-ACTH antibodies covalently on the nanostructured SPGE surface. The surface-immobilized anti-ACTH antibodies captured the biotinylated ACTH (biotin-ACTH) and non-labelled ACTH for the competitive immunoassay. After coupling of a streptavidin-alkaline phosphatase conjugate (Streptavidin-ALP), the bio-catalysed precipitation of an insoluble and insulating product onto the sensing interface changed the charge transfer resistance (R_ct) characteristics significantly. The detection limit of 100 fg/mL was determined for ACTH in a 5 μL sample volume, which indicated that this versatile platform can be easily adapted for miniaturized electrochemical immunosensing of cancer marker biomolecules. High selectivity and sensitivity of our immunoassay to detect ACTH in real samples demonstrated its promising potential for future development and applications using clinical samples.