Pillar[6]arene@AuNPs Functionalized N‑CQDs@Co₃O₄ Hybrid Composite for Ultrasensitive Electrochemical Detection of Human Epididymis Protein 4

Hydroxylatopillar­[6]­arene (HP6) has been used as a stable ligand for the synthesis of gold nanoparticles (AuNPs). HP6 with excellent molecular recognition capability will securely capture the electron transfer medium thionin (Thi). Compared with other electroactive molecular capture methods (physical adsorption, etc.), the formation of an HP6⊃Thi complex can effectively inhibit the leakage of Thi and significantly increase the loading of Thi. HP6@Au was prepared in an aqueous solution with OH^– at room temperature through an oxidation–reduction reaction between HP6 and AuCl₄^–, without the need for conventional harsh reducing agents. X-ray photoelectron spectroscopy and Fourier transform infrared studies have shown that the −OH groups in HP6 are oxidized to −COOH groups, and Au³⁺ in AuCl₄^– is reduced to Au⁰, leading to nucleation and production of AuNPs. The formation of HP6@Au strengthens the electron transfer rate, increases surface area, and captures a large number of primary antibodies (Ab1). Besides, the surface of N-doped carbon quantum dots and cobalt oxide (N-CQDs@Co₃O₄) can be used for the deposition of HP6@Au by π–π interaction to form a green highly conductive nanocomposite. An electrochemical immunosensor with significant stability was constructed based on HP6@Au/N-CQDs@Co₃O₄, and it was used for the effective detection of human epididymis protein 4 (HE4). The detection limit of the proposed immunosensor platform is 1.5 pg mL^–1, and it provides a wide linear range from 0.002 to 20 ng mL^–1 under optimal conditions. The analysis results of HE4 in human serum are satisfactory and have broad application prospects in clinical monitoring.