Aptamer Based Stress Detection Biosensor
Authors: Uchida Rina , Sugimoto Mai , Mitsunaga Yasushi , Torisawa Shinsuke , Kobayashi Yasuhisa , Saiki Tsunemasa , Takamura Eiichiro , Sakamoto Hiroaki 
Published: December 2024

Depression is one of the most common mental disorders, and stress is a major factor behind the occurrence of depressive symptoms. Stress refers to the internal reaction of the body to an external stimulus, and the external stimulus that causes stress is called a stressor. Stressors can be broadly classified into physical and chemical stressors, physiological stressors, and psychological and social stressors. Among these, humans are particularly affected by psychological and social stressors. Therefore, it is very important to accurately assess the level of psychological and social stress in daily life in order to maintain mental health. Currently commonly used stress diagnostic methods such as electroencephalography (EEG) and electrocardiography (ECG) are relatively complex and are not suitable for personal use in public places or for point-of-care testing (POCT) because of the large size of the EEG and ECG equipment used to perform the tests. Therefore, attention has focused on methods to detect stress-related biomarkers from biofluids such as sweat and interstitial fluid. One of the most well-known stress-related biomarkers is cortisol. Cortisol is a steroid hormone with a molecular weight of 362.46 g/mol and is one of the major glycocorticoids synthesized in the bundle zone of the adrenal cortex. Cortisol levels fluctuate rapidly throughout the day. When humans are exposed to various psychological and physiological stresses, cortisol levels fluctuate rapidly throughout the day. Therefore, there is a need to establish a technique to detect cortisol in situ under stress-inducing conditions. In this study, we aim to construct an electrochemical biosensor using aptamers with pseudoknot structures as molecular recognition elements, which have the advantages of high sensitivity, short detection time, easy miniaturization, and low cost. A pseudoknot structure is a secondary structure of nucleic acid containing two stem-loop structures such that one side of one stem is located between the other stems. While the sensors reported so far allow for convenient detection of the target, the structure of the aptamer in the absence of the target is not defined and the distance between the mediator and the electrode is not controlled. Therefore, the sensitivity is compromised by high background current and low electron transfer efficiency. However, by applying the pseudoknot structure, it is possible to dichotomize the structure in the absence and presence of cortisol. This makes it possible to reduce the background current and is expected to increase the sensitivity of the sensor. In this sensor, an aptamer is immobilized on a gold electrode via an Au-S bond and modified with a mediator at the end. When cortisol, the target, is then added, the aptamer recognizes cortisol and binds specifically to it. This changes the structure of the aptamer and brings the mediator closer to the electrode surface. Therefore, as the concentration of cortisol increases, an increase in current is observed due to the increased electron transfer efficiency of the mediator, and detection is performed by observing this change in current value (Fig. 1). The purpose of this study is to develop a biosensor that can comprehensively diagnose stress levels in humans using this technique. The experimental procedure first involved heat treatment of the aptamer to control its conformation in the absence of the target before immobilizing the aptamer on the gold electrode of the sensor. Then, 10 µl drops of aptamer were immobilized overnight followed by washing. Then, a drop of ferrocene carboxylic acid solution, a mediator, was added to the electrode to modify the aptamer ends with ferrocene. Cyclic voltammetry (CV) was performed using the prepared electrode to measure cortisol. As a result, a reduction peak around +0.56 V was observed. This peak is thought to be derived from the ferrocenecarboxylic acid modified at the end of the aptamer. An increase in the reduction peak was observed with increasing cortisol concentration. This suggests that there is a strong correlation between cortisol concentration and the reduction current value. Since the normal value of cortisol concentration in humans is approximately 166 nM to 635 nM, it is clear that this sensor covers the human cortisol concentration range.

Figure 1