Smartphone-Coupled Dual-Signal Biosensor Based on Au NP–Chitosan Hydrogel: Colorimetric Readout and Sol–Gel Transition for On-Site Cysteine Monitoring

Cysteine (Cys) is essential in human physiological processes, and its abnormal levels are linked to various diseases, necessitating real-time monitoring in biological samples. Conventional detection methods, however, are often limited by the operational complexity and susceptibility to interference. To overcome these issues, we developed a chitosan–gold nanoparticle (CS–Au NP) gel sensor for dual-mode visual detection of Cys via gel–sol transition and colorimetry. This approach significantly reduces false positives that are common in single-signal methods. The sensor utilizes chitosan as an in situ reductant to form Au NPs, which together with chitosan amine groups and hydrogen bonding construct a 3D gel network. In the presence of Cys, its thiol groups competitively bind to Au NPs, disrupting cross-linking and triggering a gel–sol transition. Quantitative analysis based on the coverage ratio (Cr) on paper substrates showed high sensitivity with a detection limit of 2.08 μM and strong linearity ( R 2 = 0.9981). Concurrently, Au NPs released from the gel assemble into nanoaggregates, causing a red-to-blue-gray color shift due to SPR changes. This allows both visual qualitative assessment and smartphone-based RGB quantification, achieving a linear range of 20–1000 μM and an LOD of 2.23 μM. The dual-mode design ensures high selectivity, with minimal response to 19 interfering amino acids. Practical tests in human urine and bovine serum yielded recoveries of 100.24%–125.80%, confirming accuracy in complex matrices. Integrated with smartphone imaging, this portable system is ideal for point-of-care testing, offering a reliable and cost-effective platform for Cys monitoring and precision medicine.