Salivette swab

In this study, five volunteers with good health status, and with no previous diagnosis of cardiovascular diseases, neurological or severe systemic disorders, were enrolled (average age 33.4 y/o; all subjects were female and non-smokers). According to the Helsinki Declaration, ethical approval was obtained from the Institution Ethical Committee and all recruited subjects signed informed consent forms before the collection of serum and saliva samples. Serum was obtained by the centrifugation of blood at 2500 g for 10 min at room temperature, and then stored at −80 °C before analysis. For the collection of saliva, Salivette^® swabs were purchased from Sarstedt (Sarstedt AG & CO, Numbrecht, Germany). Each Salivette^® contains a cotton swab that can filter saliva and retain any food residues. The saliva collection procedure was performed following the manufacturer’s instructions. Briefly, the swab was placed in the mouth and chewed for one minute, at least two hours after the last meal and teeth brushing. Then, the swab was centrifuged at 1000× g for 5 min in order to recover the biofluid. Saliva samples were stored at −20 °C before analysis. This method was previously reported for the collection of saliva for Raman analysis, demonstrating no interference in the subsequent spectroscopic evaluation [6 (link),7 (link),8 (link),9 (link)].

Salivary cortisol, a reliable measure of HPA axis reactivity, was collected using Salivette swabs (Sarstedt^®, Nümbrecht-Rommelsdorf, Germany). Measurements occurred at five time points throughout the experiment: −60, −20, +12, +50, +60 min, relative to stress or control treatment onset. Samples were kept on ice during the session, centrifuged at 3,000 rpm at 4°C for 10 min directly following the experimental task, and stored at −70°C. Cortisol concentrations were analyzed via a commercially available ELISA kit (Salimetrics), following the manufacturer’s instructions. Inter-assay coefficient of variation ranged from 6.1 (high control) to 7.5% (low control), and intra-assay coefficient of variation was 5.3%. Changes in cortisol levels were calculated using Area Under the Curve with respect to increase (AUCi), following the procedures described by Pruessner et al. (2003) (link). Note that the Pruessner et al. (2003) (link) paper describes two methods for assessing cortisol reactivity, AUCi and AUCg (Area Under the Curve with respect to ground). We elected to use AUCi rather than AUCg because we were interested in capturing sensitivity to stress, rather than total cortisol output. However, we note that our findings are not dependent on this decision and our robust to either measure.

Blood samples for measurement of cortisol, ACTH, prolactin, TSH, and free T4 concentrations were collected between 8 and 9 AM. Standard clinical chemistry testing and estimation of ferritin levels were also performed.
Adrenocortical reserve was assessed by low-dose ACTH testing (1 μg Synacthen, Sigma-Tau Industrie Riunite, Rome, Italy). Blood and saliva samples were collected prior to testing and 15, 30, 60, and 90 min after ACTH i.v. bolus. Saliva was collected into Salivette swabs (Sarstedt AG & Co., Nümbrecht, Germany). Peak serum cortisol levels below 500 nmol/l (18 μg/dl) were considered indicative of reduced adrenocortical reserve [11 (link)].
Quality of life (QoL) was established by Short Form-36 Health Survey (SF-36). The questionnaire has been validated by standard methodology of International Quality of Life Assessment [12 (link)].

All the materials were purchased from Sigma-Aldrich (United States) and used as received if not differently specified. Salivette^® swabs for the saliva collection were purchased from Sarstedt (Germany, catalog number S1 1534). Mini Bin aluminum foils (Sigma-Aldrich, United States, catalog number Z691569-1EA) were used as Raman substrate. All the materials were used following the manufacturer’s instructions without further purification steps. All the described procedures were performed in accordance with relevant guidelines, regulations, and ethical standards. The procedures were approved by the Ethical Committee of the institution in which the experiments were done or in accord with the Helsinki Declaration of 1975.

Unstimulated whole mixed saliva was collected using Salivette swabs (Sarstedt, Nümbrecht, Germany), per the manufacturer’s instructions. All patients were provided with written instructions for the saliva sampling. Samples were collected over a 64-h period at 2-3-h intervals starting at 08:00 (Figure 1). Following centrifugation, the samples of clear saliva were stored at minus 80°C until assayed in batch (9 (link)).

Saliva collection was carried out using Salivette swabs (Sarstedt, Germany), following manufacturer’s instructions. Briefly, the swab was placed in the subject’s mouth and chewed for 60 s to stimulate salivation. The cotton swab was then closed into the provided plastic tube and centrifuged for 2 min at 1000g in order to remove eventual food debris and to collect saliva. Saliva samples were stored at − 20 °C. To limit the variability in biomolecules concentration in saliva, the collection procedure was performed at fixed time-points after breeding and teeth brushing. Pre-analytical parameters, including collection time, time between the collection of saliva and the Raman analysis and storage temperature, were recorded. All the data were anonymized preserving the subject privacy. A saliva drop (3 µl) was deposited on a glass slide covered with commercially available Aluminum foil, in order to achieve the SERS effect, and dried at room temperature for 10–15 min. Aluminum foil was purchased from Merck KGaA (Germany) and used as received to cover the glass substrate for the SERS analysis.