Development of a Prototype Lateral Flow Immunoassay of Cortisol in Saliva for Daily Monitoring of Stress

Abstract

Emotional stress negatively affects the quality of a person's daily life. From a physiological point of view, stress is expressed in the excitation of the hypothalamic-pituitary-adrenal cortex axis, which leads to the release of the hormone cortisol into the blood. We developed a lateral flow immunoassay to detect cortisol in human salivary fluid and tested it on 10 healthy volunteers daily for about one month (n = 293 saliva samples). Cortisol was detected in concentrations ranging from 1 to 70 ng/mL. Salivary cortisol levels were confirmed by ELISA. The straightness range of LFIA calibration was from 1 to 100 ng/mL. The diagnostic sensitivity of the method was 73%. It was found that in 3 out of 10 subjects, fluctuations in the level of cortisol in saliva partially corresponded to the subjectively assessed level of stress.

Keywords: emotional stress; gold nanoparticles conjugation; immunochromatographic test; point-of-care testing; rapid detection; salivatory cortisol.

Figure A1

TEM image and size distribution of synthesized Au nanoparticles.

Figure 1

Scheme of immunochromatographic analysis in a competitive format. On the (left), the analysis procedure is shown, which includes preincubation of the sample with the conjugate, as well as the scheme for applying the reagents to the test strip. On the (right), two extreme cases for the analysis result are shown: the absence of antigens in the sample, and a large quantity of antigens.

Figure 2

Calibration curve of the immunochromatographic analysis (a). A part of the calibration curve in the concentration range from 1 to 100 ng/mL was approximated by a polynomial function (b). The abscissa shows the concentration of cortisol (a) or log of the cortisol concentration (b) in the calibration solution and the ordinate shows the ratio of T to C. Error bars indicate the standard deviations (n = 3).

Figure 3

An example of test strips after examining saliva samples from study participant no. 7. It is clearly seen that the intensity of staining is different not only in the upper, but also in the lower spot.

Figure 4

Control experiment with saliva components. C1—test performed with the HSA solution (3 mg/mL), and C2—test performed with the human immunoglobulin solution (1 mg/mL). For comparison, on the right there is a test carried out with saliva as the analyzed liquid. The test was carried out simultaneously with the control tests.

Figure 5

Profiles of changes in the concentration of cortisol in saliva sampled in the morning immediately after waking up for a month for each study participant.

Figure 6

Profiles of changes in stress of each of the participants during the month. Blue denotes “cortisol” stress values expressed in C/C_max, where C is the current value of the concentration of cortisol for each study participant, ng/mL; C_max is the maximum value of the concentration of cortisol in saliva for the study period for each participant, ng/mL. Red denotes emotional stress valuesexpressed in S/S_max, where S is the current value of the emotional stress for each study participant, a.u.; S_max is the maximum value of the emotional stress for the study period for each participant, a.u.

Figure 7

Calibration graph of the enzyme immunoassay (n = 3). Curve fitting equation: ln(Y) = −0.4637290342 × ln(X) − 0.2250345633. Correlation coefficient R = 0.93.