A bio-conjugated 2D photonic crystal hydrogel-based immunoassay for glycocholic acid.

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide, underscoring an urgent need for reliable point-of-care testing (POCT) tools. Glycocholic acid (GCA), owing to its high specificity and sensitivity, has been recognised as a promising biomarker for HCC. Nevertheless, current GCA detection methodologies still suffer from drawbacks, including high cost, cumbersome procedures, prolonged assay times, and single-use formats. In this study, a novel label-free immunoassay based on a two-dimensional photonic crystal hydrogel (2D-PCH) has been reported, which enabled visual, quantitative, and reusable detection of GCA. Unlike conventional direct-detection strategies, the developed 2D-PCH employed antigen-antibody complexes as reversible non-covalent crosslinkers within the hydrogel network. Competitive binding between free GCA and the grafted antigen for the limited antibody sites disrupted these crosslinks, reducing the crosslinking density and triggering hydrogel swelling. This volumetric expansion increased the particle spacing of the embedded photonic crystal array, which was quantitatively reflected by an increase in the Debye diffraction ring diameter, accompanied by a visible structural colour shift. The 2D-PCH sensor achieved a limit of detection (LoD) of 3.66 µg mL-1 and a linear detection range spanning from 10 to 50 µg mL-1. Moreover, the developed 2D-PCH displayed excellent specificity, good reversibility and satisfactory recovery ranging from 93.02% to 106.07% towards spiked samples. Therefore, the developed 2D-PCH-based immunoassay provided a visual, label-free, convenient, quantitative and reusable POCT method for the biomarker GCA, with great potential for future HCC clinical screening applications.