Enhancing cortisol sensor performance using a heterobifunctional cross-linker with a flexible spacer arm

In today’s technology-driven, fast-paced lifestyle, stress has increasingly become an inevitable part of daily life. This has led to numerous physiological and psychological challenges for the medical research fraternity. Therefore, there is a pressing need for accurate and precise biochemical profiling of stress-signaling hormonal fluctuations. Among these biomarkers, cortisol, a steroid hormone closely linked to stress, and nanosensors for cortisol have been widely reported. In this work, A graphene-based electrode sensor was developed for the selective detection of cortisol, utilizing two distinct cross-linking chemistries: the conventional zero-length EDC–NHS linker and the bifunctional N-k-maleimidoundecanoyl-oxysulfosuccinimide ester (SKMUS), which features a 16.3 Å hydrocarbon spacer arm. The study aimed to investigate the effect of linker length and architecture on the surface accessibility and functional activity of a cortisol-specific aptamer. The surface coverage of SKMUS on the graphene oxide-modified screen-printed electrode (GO-SPE) and the aptamer density immobilized via SKMUS chemistry, when compared with that of EDC-NHS chemistry, provided insights into the steric effects and the role of linker length on the sensitivity of cortisol sensing. Furthermore, Atomic Force Microscopy (AFM) provided a detailed, stepwise surface analysis throughout the sensor fabrication process. The results indicated that SKMUS facilitated a more uniform and flexible aptamer orientation due to its optimal spacer length, enhancing target accessibility and recognition efficiency. High specificity was ensured by a 14-mer, high-affinity, rationally truncated aptamer (derived from the 61-mer parent sequence) that minimized cross-reactivity with structural analogues of cortisol. These enhancements in sensitivity, achieved through cross-linking chemistry, can enable early diagnosis, improved monitoring, and preventive therapeutics, paving the way for more effective stress management strategies.