Glucose solution

For ITT and GTT assays, after a 6 h fasting period, rats were injected intraperitoneally with a saline injection of insulin (0.75 U/kg BW of Novo Rapid, Novo Nordisk^®, Paço de Arcos, Portugal), or with a glucose solution (2.0 g/kg BW, Sigma-Aldrich, Merck, St. Louis, MO, USA, EUA), respectively. Blood samples were collected from the tail vein, being the first drop of blood discarded, and the second drop used to measure baseline glucose levels. Serum glucose measurements occurred at subsequent time points following glucose or insulin administration (ITT assay: 15, 30, 45, 60, 120 min; GTT assay: 15, 30, 60, 120 min) at selected time-points. The glucose levels were measured using a portable commercial glucometer kit (GlucoMen^® aero 2K, A. Menarini Diagnósticos, Paço de Arcos, Portugal). The area under curve (AUC) of ITT and of GTT curves were calculated using the trapezoidal method [18 (link),19 (link)].

Mice were fasted for 16 h before oral glucose tolerance tests (OGTTs) after 6 weeks of dietary manipulations. Glucose solution (3 g/kg; Sigma Aldrich, St. Louis, MO, USA) was administered to WT and AD mice by oral gavage. Blood glucose was measured using a glucometer (Bioptik Technology, Taipei, Taiwan).

Before starting the measurement, the electrodes are preconditioned in 10 mM PBS (pH 7.4) to ensure same measurement conditions in all WEs for sensitive and reproducible results and to electrochemically remove possible residues from the electrodes. The measurement itself is also started in 10 mM PBS until the signal saturates, then it is changed to a 40-mM glucose solution (40 mM in 10 mM PBS (Sigma Aldrich, USA), substrate for the glucose oxidase (GOx) enzyme used). 2 - and 5-min stop-flow protocols are applied to achieve a signal amplification (Fig. S5). During the stop phase, hydrogen peroxide (H₂O₂) produced by the enzymatic reaction is accumulated in the channel, which is then flushed over the electrochemical cell when restarting the flow, to generate the typical rectangular-like current peaks for stop-flow measurements [35 (link)]. In the case of multiplexed biosensors, the primary peaks correspond to the accumulation of electrochemical active species in the immobilization area during the stop phase. During the “flow” phase, these species are also passing through neighboring electrochemical cells, in addition to their own individual electrochemical cell, which results in the following current peaks.