Having a generalizable point of care (POC) method for clinically relevant biomolecules would greatly enhance healthcare management and disease diagnosis. Recently our group developed a novel DNA nanostructure architecture for versatile detection of analytes which is generalizable for assaying several small molecules and their larger protein binding partners (e.g. antibodies) in human serum. The DNA nanostructure is built through on-electrode enzymatic ligation of three oligos for electrode attachment, anchor binding, and electrochemical signaling. In this study, we developed an economical synthetic approach for making oligonucleotide-steroid conjugates, and we explored the capability of these DNA nanostructure sensors for small molecule/steroid detection.

Cortisol is a steroid hormone secreted by the hypothalamic-pituitary-adrenal system in response to the body’s stress level. As the main stress hormone, it controls processes such as immune, adrenal, circulatory, and metabolic. Moreover, anomalies of cortisol levels can result in serious conditions such as Addison’s disease, Cushing’s syndrome, and adrenal insufficiencies. Every year, 120,000 deaths are attributed, in part, to elevated levels of stress. To minimize these issues, a simplified method for cortisol sensing is vital. In this study, we conjugated cortisol to amine-tagged DNA and used the conjugates in our sensors. Anti-cortisol antibodies induced a 67% signal drop, validating conjugation. A calibration curve for cortisol showed a limit of detection of 800 pM and a dynamic range of 1 – 100 nM. The sensor was validated in saliva and human serum samples using the gold standard method, ELISA. Changing cortisol levels in two human patients' saliva samples were successfully detected after several collection times throughout a single day. As a potential point-of-care (POC) detection device, our novel cortisol biosensor could detect the hormone in human serum and saliva samples within 6 minutes.