An Efficient Bio-Receptor Layer Combined with a Plasmonic Plastic Optical Fiber Probe for Cortisol Detection in Saliva

Abstract

Cortisol is a clinically validated stress biomarker that takes part in many physiological and psychological functions related to the body's response to stress factors. In particular, it has emerged as a pivotal tool for understanding stress levels and overall well-being. Usually, in clinics, cortisol levels are monitored in blood or urine, but significant changes are also registered in sweat and saliva. In this work, a surface plasmon resonance probe based on a D-shaped plastic optical fiber was functionalized with a glucocorticoid receptor exploited as a highly efficient bioreceptor specific to cortisol. The developed plastic optical fiber biosensor was tested for cortisol detection in buffer and artificial saliva. The biosensor response showed very good selectivity towards other hormones and a detection limit of about 59 fM and 96 fM in phosphate saline buffer and artificial saliva, respectively. The obtained detection limit, with a rapid detection time (about 5 min) and a low-cost sensor system, paved the way for determining the cortisol concentration in saliva samples without any extraction process or sample pretreatment via a point-of-care test.

Keywords: biosensor; cortisol; glucocorticoid receptor (GR); optical sensors; plastic optical fibers (POFs); point-of-care test (POCT); stress; surface plasmon resonance (SPR).

Figure 1

Scheme of the experimental setup employed to test the GR-SPR-POF biosensor.

Figure 2

(A) Scheme of the functionalization protocol used. (B) SPR spectra attained by using PBS as bulk solution after each step of the immobilization procedure. (C) Variation in resonance wavelength (Δλ) computed with respect to the SPR wavelength obtained on the non-functionalized chip.

Figure 3

GR–cortisol binding tests in PBS. (A) SPR spectra, smoothed and translated along the y-axis direction, obtained in PBS at increasing cortisol concentrations. (B) Result of the signal processing performed to determine the resonance wavelengths useful to obtain the dose–response curve in PBS.

Figure 4

GR–cortisol binding test in artificial saliva diluted 1:50 with PBS. (A) SPR spectra obtained in diluted artificial saliva at increasing cortisol concentrations. (B) Result of the signal processing performed to determine the resonance wavelength useful to obtain the dose–response curve in artificial saliva (diluted 1:50).

Figure 5

Dose–response curves obtained through cortisol monitoring in (A) PBS and (B) artificial saliva diluted 1:50. The Δλ absolute values (computed in relation to the blank) as a function of increasing cortisol concentrations on the GR-SPR-POF platform are reported in a semi-log scale, along with Langmuir fitting of the experimental data.

Figure 6

(A) Resonance wavelength variation for the structural analogues of cortisol (estradiol 100 pM and progesterone 100 pM) and cortisol (10 pM). One-way ANOVA * p < 0.01 vs. estradiol and ^# p < 0.01 vs. progesterone. (B) Comparison between the resonance wavelength variation achieved by a solution with cortisol only (10 pM) and with cortisol pooled in a mixture with progesterone and estradiol, each of which was considered at a concentration of 10 pM.