Colorimetric analysis, which relies on a chemical reaction to facilitate a change in visible color, is a great strategy for detecting cortisol, which is necessary to diagnose and manage the wide variety of diseases related to the hormone, because it is simple in design, inexpensive, and reliable as a standard cortisol analysis technique. In this study, four different colorimetric cortisol analyses that use various chromogens, which include sulfuric acid, Porter-Silber reagent, Prussian blue, and blue tetrazolium, are studied. Modifications to the classic Porter-Silber method are made by increasing the carbon content of the alcohol and adding gold nanoparticles, which result in a twofold increase in reaction rate and a slight decrease in the limit of detection (LoD). After a comparison of the reaction rate, LoD, dynamic range, characteristic peaks, and color stability of all methods, blue tetrazolium demonstrates a low LoD (97 ng/mL), broad dynamic range (0.05-2 μg/mL), and quick reaction rate (color development as fast as 10 min), which are well within the requirements for human biofluids. Cortisol in artificial saliva and sweat and in human sweat was determined while confirming that no excipients or other biomarkers interfered with the reactions. Twenty-one human sweat samples were tested using blue tetrazolium and revealed a significant difference between male and female apocrine cortisol concentrations and showed a highly significant difference between apocrine and eccrine cortisol concentrations. Colorimetric methods of cortisol can compete with existing electrochemical sensors because of their similar accuracy and detection range in certain wearable biosensor applications. The simplicity of colorimetric methods advances potential applications in skin-interfaced bio-electronics and point-of-care devices.
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