Fabrication of an Electrochemical Aptasensor for Stress Hormone Using a Rationally Truncated Aptamer on Graphene Quantum Dots Modified Screen Printed Electrodes

Abstract

Nowadays, stress is one of the major causes of pathologies in the human body. In India, most of the time around 87% of women feels stressed. World Health Organization reported that globally 450 million people suffer from mental health related problems while in India the morbidities due to mental health showed an increasing trend from 9.5 to 102.8 per 1000 persons. Current techniques used to measure stress levels consist of self-reporting method and multimodal physiological analysis. Self-reporting consists of physical interviews and self-response questionnaire. Stress-response questionnaire lacks in direct link to stress response while physical interviews are time intensive process and also require trained interviewees. Furthermore, these cannot be used for the continuous monitoring. Multimodal physiological analysis involves the monitoring of the heart rate variability, blood pressure; brain activity and skin conductance. Major advantage in using multimodal physiological analysis is the non-invasive measurements. Still, continuous monitoring is challenging and lacking direct link with the stress. Since stress varies with time, it is also difficult to quantify it with these techniques as there is no standard available. For the monitoring of stress, the monitoring of change in concentration in biochemical marker in biofluids is the need of an hour. Cortisol, a steroid hormone and major glucocorticoid in human body is the key biomarker for stress and hence also known as stress hormone. There are many techniques to detect cortisol such as Enzyme Linked Immuno Assay (ELISA), Electro Chemi Luminescence Immuno Assay (ECLIA), chromatography and other immunoassays but these techniques are limited only up to the laboratory levels, required large volume of samples and skilled personnels. Hence to overcome these limitations, Biosensor came into picture. In biosensor, various types of bio-receptors have been used for the sensing like antibody, aptamer and molecularly imprinted polymers (MIP) etc. Due to more stability at room temperature and more specificity, aptamers are preferred as bio-receptor over antibody and MIP. Aptamers are RNA or ssDNA molecules generated by an in vitro evolution process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). For cortisol, a 61-mer long sequence of aptamer has been generated and used for sensing but in principle only a portion of aptamer participates in its interaction with the analyte. Hence in the present work, we have rationally truncated long sequence of aptamer into a smaller variant of 14-mer by considering the secondary structure of the aptamer. Since nanomaterials are the suitable candidates for the immobilization of the bioreceptor. Hence, we have synthesized and characterized graphene quantum dots(GQDs) using various characterization such as UV, PL, FTIR and RAMAN. For the electrochemical sensing of cortisol, Aptamers were immobilized on the GQDs modified electrodes. Performance of both the aptamers in terms of binding affinity, limit of detection and specificity were compared. Both the aptamers exhibited same limit of detections of 0.1 pg/ml but in terms of specificity 14-mer aptamer showed better results than the parent 61-mer aptamer. Parent aptamer showed interferences with the structural analogues of the cortisol i.e. cortisone, corticosterone and triamcinolone while truncated aptamer showed almost negligible interferences with cortisone and triamcinolone. Also, on truncation of the aptamer, binding affinity was improved by ~1900 fold which indicate the superiority of truncated aptamer (14-mer) over the parent aptamer (61-mer).