Ultrasensitive Detection of Hair Cortisol Based on Portable Raman Spectrometer and Double-layer Paper Microdevice

Cortisol in the human hair is a clinical biomarker of long-term social-and-work-related mental stress, which is of high morbidity rate in the current modern society. This study developed a sensitive hair cortisol assay, featuring surface- enhanced Raman spectroscopy, immunoreaction, and a double-layer paper microdevice. The first layer of the paper microdevice was used to remove the hair residue in the hair extract by filtration (sample pretreatment). The second layer was used for competitive immunoreaction and detection. Standard cortisol antigen immobilized in the second layer and the free cortisol in hair extract competed to bind the spiked Raman-active cortisol monoclonal antibody solution. The hair cortisol can be quantitated by the intensity of Raman signal of monoclonal antibody bound on the paper. We found the Raman signal decreased as the cortisol concentration increases in hair samples. The relative Raman intensity measured was linearly proportional to the logarithmic value of the cortisol concentration in hair samples we measured. The detection of limit (LOD) was 1 pg/mL with the portable Raman spectrometer. The RSD of measurement was 8.38% (n=6). In addition, we used LC-MS to measure two real samples as a comparison with our method as above. The results are 0.771 and 0.153 ng/mL by LC-MS method and 0.63 and 0.247 ng/mL by the proposed method. It can be observed that the results are in same order, demonstrating the validity of the proposed method. In addition, 48 samples can be measured in a single chip. These results showed that this method is sensitive, specific, and suitable for large-scale screening of hair cortisol samples.