Nanoquant

Maternal blood samples were centrifuged at 10.000 g for 20 min at 4°C and the supernatant plasma was immediately frozen at -20°C. For progesterone analysis, plasma samples were diluted 1:200 using ELISA Buffer and measured with a competitive immunoassay (Progesterone ELISA Kit, Cayman Chemical, Michigan, USA) on a NanoQuant (Tecan Group AG, Männedorf, Switzerland) according to manufacturer’s instructions.

Maternal blood samples were centrifuged at 10,000 g for 20 min at 4°C and the supernatant plasma was immediately frozen at −20°C. For progesterone analysis, plasma samples were diluted 1:200 using ELISA Buffer and measured with a competitive immunoassay (Progesterone ELISA Kit, Cayman Chemical, Michigan, USA) on a NanoQuant (Tecan Group AG, Männedorf, Switzerland) according to manufacturer's instructions.

Total RNA was isolated from about 1 million fibroblasts or cancer cells using the RNeasy Mini Kit (cat. #74104, Qiagen) following the manufacturer’s instructions, including a DNase I treatment step (cat. #79254, Qiagen). RNA from the column was eluted with 30 µl of RNase-free water (from the kit).
Photometrical adsorption measurements at 280 nm and 260 nm confirmed purity and quantity of the purified DNase-treated RNA (NanoQuant, Tecan).

Mothers were asked to take three samples of their children’s saliva on one weekday of the week during which they wore the actigraphy watch. An oral fluid collector (OFC) consisting of a synthetic polymer swab designed to collect 0.5 mL of saliva mixed with 3 mL of OFC buffer was used to collect the samples (Soma Bioscience Ltd., Wallingford, UK). With this technique, samples are stable at room temperature for several weeks and are unaffected by recent food and drink ingestion. Salivary cortisol was determined using an enzyme immunoassay (EIA) test kit (Soma Bioscience Ltd., Wallingford, UK) and read by an automated analyser (Tecan Nanoquant, Männedorf, Switzerland). The assay range for cortisol was 0.25–32.0 ng/mL. In the case where maximum values were obtained, samples were titrated and re-analysed. Cortisol profiles were assessed using all 3 values to determine if each participant had a flat or normal cortisol profile compared to the individual group mean.
Each participant was required to provide salivary samples for 3 time points over a 24-h period. The timings were: (a) afternoon at approximately 4 p.m. (baseline); (b) 30 min before habitual bedtime; (c) 30 min after waking. Either parents or participants themselves (under adult supervision) were asked to place a cotton/polymer swab in their mouth for 30–45 s in order to collect each sample.

The production of H₂O₂ by BSA-GOx-NPs, glucose, and oxygen was determined via a classic colorimetric method, applying ammonium titanyl oxalate as the indicator. In brief, 0.1 mL of BSA-GOx-NPs or BSA-NPs suspension (50 μg/mL BSA) was mixed with 0.1 mL of different glucose concentrations (0, 0.025, 0.05, 0.1, 0.2, 0.5, 1 and 2 mg/mL). After 1 h, ammonium titanyl oxalate solution (10 μL, 10 mM) was added. The obtained yellow suspension was measured by a microplate reader (Nano Quant, Tecan) at 405 nm.