Anhydrous glucose

Alginic acid sodium salt from brown algae (BioReagent, Sigma-Aldrich, St. Louis, MI, USA), glucose oxidase from Aspergillus niger (type II, ≥15,000 U/g solid, Sigma-Aldrich, St. Louis, MI, USA), nickel(II) chloride (NiCl₂·6H₂O) Merck KGaA, Darmstadt, Germany), iron(III) chloride (FeCl₃·6H₂O) (Merck KGaA, Darmstadt, Germany), glucose anhydrous (≥98.0%) (Sigma), acetic acid (CH₃COOH) (Merck KGaA, Darmstadt, Germany), hydrogen peroxide (H₂O₂) 30% (Merck KGaA, Darmstadt, Germany), sodium hydroxide (NaOH) (Merck KGaA, Darmstadt, Germany), disodium hydrogen phosphate (Merck KGaA, Darmstadt, Germany) and sodium dihydrogen phosphate (Merck KGaA, Darmstadt, Germany).

The NC membrane was obtained from mdi Membrane Technologies (Harrisburg, PA, USA). Gold chloride hydrate (HAuCl₄), trisodium citrate, Triton X-100, Tween-20, L-arginine, polyvinyl alcohol (PVA, 9000–10,000 Da), glucose anhydrous, sucrose, albumin from human serum, and goat anti-mouse IgG antibody (capture antibody II) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Alexa Fluor^® 555-labeled rabbit anti-mouse antibody was obtained from Invitrogen (Carlsbad, CA, USA). Sodium chloride was purchased from Duchefa Biochemie (BH, Haarlem, Netherlands). Bovine serum albumin (BSA) was obtained from BioWorld (Dublin, OH, USA). Polyethylene glycol (PEG, 3400 Da) was obtained from Polyscience (Warrington, PA, USA). The mouse anti-human microalbumin antibody (clone number: 15C7, detection antibody) and mouse anti-human microalbumin antibody (clone number: 1A9, capture antibody I) were purchased from Hytest (Turku, Finland). Artificial urine was purchased from Pickering Laboratories (Mountain View, CA, USA). For the reaction solutions, 50 mM PBS buffer (pH 7.2), 0.1 M PBS buffer (pH 7.2), PBSB buffer (PBS buffer and 1% BSA), running buffer (1% BSA, 1% sucrose, 50 mM NaCl, 50 mM L-arginine, 0.2% PEG, 0.5% PVA, 0.2% Tx-100, and 0.2% Tween-20), and double distilled and deionized water (DDW) were prepared.

The IPCs were assessed the function by glucose-stimulated C-peptide secretion assay (GSCS)^{24 (link),38 (link),69 (link),70 (link),138 (link)}. The IPCs were incubated in normal KRH bicarbonate (KRBH) buffer (pH 7.4) for 1 h as basal control, then in 5.5 mM of glucose anhydrous (Sigma) in KRBH for the next 1 h and finally in 22 mM glucose anhydrous in KRBH for 1 h. Enzyme-linked immunosorbent assay (ELISA) kit (Millipore) was used for detecting the generated C-peptide level according to the manufacturer’s protocol. Total DNA (ng) and stimulation time (mins) were used to normalization.

Streptozotocin (Merck Millipore, Temecula, CA, USA), nicotinamide, sodium citrate monohydrate, phosphate-buffered saline (PBS) and glucose anhydrous (Sigma-Aldrich, St. Louis, MO, USA). Citric acid (Merck, Kenilworth, NJ, USA), glibenclamide (Sigma-Aldrich, USA), formaldehyde (HmbG chemicals, Hamburg, Germany), one-touch glucometer (Accu-Chek Performa, Roche, Mannheim, Germany). Rat Insulin ELISA Kit (ER1113, Wuhan Fine Biological Technology Co., Ltd., China). 2,4,6-Tri (2- pyridyl)-s-triazine, Folin-Ciocalteu reagent and ferric chloride (Sigma-Aldrich, USA). Cellulose extraction thimble (Tokyo Roshi Kaisha, Ltd, Tokyo, Japan), ferrous sulphate heptahydrate (Essex, UK). Amylase and glucosidase enzymes, 3, 5-dinitrosalicylic acid (DNSA) and 4-Nitrophenyl (3-D-glucopyranoside (_P-NPG) substrate, monobasic potassium phosphate and potassium tartrate tetrahydrate (Sigma-Aldrich, USA), α-acarbose 95% (Acros organic, USA). Potato starch (Sigma-Aldrich, USA).

N-isopropylacrylamide (NIPAAm) 97%, N,N′-methylenebisacrylamide (MBA) 99%, N,N,N′,N′-tetramethylethylenediamine (TEMED) 99%, potassium persulfate (KPS) 99%, poly(methyl vinyl ether-alt-maleic anhydride) (PVME-MA) 216,000 g mol⁻¹, sodium dihydrogen phosphate 99%, disodium hydrogen phosphate 99%, citric acid 99.5%, potassium hydroxide 85%, calcium hydroxide 95%, bovine serum albumin 96%, lactic acid 85%, acetic acid 99.7%, glycerol 86%, urea 98%, glucose anhydrous 96%, and metronidazole (MTZ) 98% were purchased from Sigma-Aldrich. Sodium chloride 99% was obtained from Meyer. All reagents were of analytical grade and used as received without further purification. The aqueous solutions were prepared with deionized water, purified by a Milli-Q Organex system (Millipore, Molsheim, France).

Carbohydrate analysis was carried out on a Shimadzu (Kyoto, Japan) HPLC system which consisted of an LC-20AD prominence solvent delivery module, a DGU-20A5R degassing unit, an SIL-10 AF automatic sample injector, and an RID-10A refractive index detector. The instrument was coupled with a computer equipped with LabSolutions Lite Version 5.52 software. The analytical column used for carbohydrate (fructose, glucose, sucrose, maltose, melezitose, raffinose and xylose) separation was an Agilent Technologies ZORBAX NH2 (Santa Clara, CA, USA) (4.6 × 250 mm, 5 μm particle size). The mobile phase consisted of HPLC-grade acetonitrile (J. T. Baker, Devnter, The Netherlands) and ultrapure water (70/30, v/v), while the operating conditions were: an injection volume of 10 µL, a mobile phase flow of 1 ml/min and a temperature of 30 °C. Carbohydrates were identified according to their retention times, and quantification was performed via external calibration carried out with carbohydrate standards suitable for HPLC analysis. Fructose, anhydrous glucose, sucrose, raffinose pentahydrate and melezitose hydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA), while xylose and maltose monohydrate were purchased from Kemika (Zagreb, Croatia) [21 ].