Citation: Semwal, V.; Højgaard, J.; Møller, E.; Bang, O.; Janting, J. Study on Cortisol Sensing Principle Based on Fluorophore and Aptamer Competitive Assay on Polymer Optical Fiber. Photonics 2023, 10, 840. https://doi.org/10.3390/ photonics10070840 Received: 15 June 2023 Revised: 11 July 2023 Accepted: 19 July 2023 Published: 20 July 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). photonics hv Communication Study on Cortisol Sensing Principle Based on Fluorophore and Aptamer Competitive Assay on Polymer Optical Fiber Vivek Semwal 1, * , Jonas Højgaard 2 , Emil Møller 2 , Ole Bang 1 and Jakob Janting 1, * 1 DTU Electro, Department of Electrical and Photonics Engineering, Technical University of Denmark, 2800 Kongens Lyngby, Denmark; oban@dtu.dk 2 Pisco Group, Rønnegade 1, 3. th., 2100 Copenhagen, Denmark * Correspondence: vivse@dtu.dk (V.S.); jajant@dtu.dk (J.J.) Abstract: In this study, we present a polymer optical fiber fluorophore/aptamer competitive assay- based cortisol sensing principle. We developed a low-cost, two-fiber perpendicular design for fluorophore-based sensing with less input light interference and high output signal intensity. The design is suitable for narrow stokes shift fluorophores. We have demonstrated the cortisol sensing principle based on the competition between tagged and normal cortisol. To date, the sensing design has exhibited a slow response, and we identified possible modifications for improvement. Our estimation shows that with miniaturization and a modified sensor assay compartment design, a less than one-hour response time can be achieved. The reported sensing principle and low-cost new design will be helpful for the future development of fluorophore-based fiber optic aptasensors that can potentially be used in a wet environment for online sensing. Keywords: fluorophore; aptamer; optical fiber; competitive assay; sensing 1. Introduction Cortisol is a stress hormone that plays a crucial role in humans and animals. In humans, it is an important biomarker for various diseases such as cardiovascular, immune, renal, skeletal, and endocrine system-based diseases [1,2]. Abnormal cortisol levels can cause irritation, depression, obesity, fatigue, bone fragility, and increased amino acid levels in the blood [2]. Cortisol can also affect, e.g., fish welfare, growth rate, and production of meat because stressed fish consume most of their energy for stress-related activities. In the literature, the best limits of detection (LOD) for cortisol are 36 fg/mL [3] and 25.9 fg/mL [4]. The LOD for chromatographic techniques [2] and ordinary immunoassays like enzyme-linked immunosorbent assays (ELISA) [5] are approximately 1 pg/mL and 50 pg/mL, respectively. The concentration range of cortisol in humans and aquaculture is μg/mL and pg/mL, respectively, and therefore a broad range of LODs are of interest [1,2]. Currently used measurement techniques require time-consuming sampling and laboratory analysis. Further, available methods for cortisol sensing are expensive and cannot be used for online monitoring. Optical fiber sensors are attractive because they have several advantages, such as the feasibility of online measurements in water over long distances [1]. The glass optical fiber cortisol sensor developed by Usha et al. [4] is of high performance, but at the expense of being bulky and complicated to fabricate, requiring operation in transmission mode and feeding/removal of an analyte. In our polymer optical fiber (POF) immunosensor design, we have used, for the first time, a simpler design comprising perpendicular POFs, a hydrogel, and a luminescent competitive assay. Aptamers are single-stranded DNA and are widely used for small molecule detec- tion in biosensors [6]. Aptamers are developed by the systematic evolution of ligands by exponential enrichment (SELEX). In this process, a multi-round in vitro process is used to isolate aptamers from pools of single-stranded oligonucleotides with randomized se- quences [7,8]. In recent years, aptamers have become the first choice for the recognition Photonics 2023, 10, 840. https://doi.org/10.3390/photonics10070840 https://www.mdpi.com/journal/photonics