Direct q

L-lactic acid (CAS: 79-3344, 129-02666), sodium chloride (CAS: 7647-14-5, 195-15975), potassium dihydrogen phosphate (CAS: 7778-77-0,164-22635), disodium hydrogen phosphate (CAS: 7558-79-4, 042-30055), urea (CAS: 57-13-6, 215-00172), ethanol (CAS: 64-17-5, 057-00456), D-glucose (CAS: 50-99-7, 049-31165), L-valine (CAS: 72-18-4, 228-00082), L-leucine (CAS: 61-90-5, 124-00852), and ammonia (CAS: 1336-21-6, 013-17505) were all purchased from Wako, Japan. Lactate oxidase (LOD) from Aerococcus viridans (T-47, Asahi Kasei Pharma, Tokyo, Japan) was purchased from Asahi Kasei Pharma and was used to modify the electrode of the biosensor. Osmium-wired horseradish peroxidase (002096, Bioanalytical Systems, West Lafayette, IN, USA) was purchased from BAS Inc., Tokyo, Japan. Phosphate-buffered saline (PBS, pH 7.4, 50 mM (PO₄)) was prepared in the laboratory. Ultrapure water obtained from the water purification system (Direct-Q, Merck, Darmstadt, Germany) was used for the preparation of all aqueous solutions.

ADA was synthesized by the oxidation of alginate using sodium (meta)periodate (NaIO₄) in a solvent consisting of water and ethanol (50:50) according to the protocol of Genc et al., with slight modifications [26 (link)]. Briefly, 1.34 g NaIO₄ were dissolved in deionized (DI, Direct-Q^®, Merck Millipore, Germany) water and added under dark conditions to an alginate dispersion consisting of 10.0 g alginate in ethanol. This reaction mixture was vigorously stirred for 6 h under light absence and subsequently quenched by the addition of 10.0 mL of ethylene glycol under continuously stirring. After 30 min, the reaction mixture was left to rest for 10 min, enabling the formation of two phases, whereas the upper aqueous phase was slowly decanted. Then, the resultant ADA suspension was dissolved in 400 mL DI water and subsequently dialyzed (MWCO: 6–8 kDa, Spectrum LAB, USA) in darkness for further 4 days with daily water exchanges and finally lyophilised.

The synthesis process of ADA-GEL hydrogel was elaborately described in our previous study [23 ]. Shortly, ADA-GEL hydrogel was synthesized by covalent crosslinking of alginate dialdehyde (ADA) and gelatin. ADA was synthesized by controlled oxidation of sodium alginate with sodium metaperiodate (oxidizing agent) in equal volume of ethanol-water mixture. After 6 h the reaction was quenched by adding ethylene glycol (VWR int., Leuven, Belgium) and dialyzed against ultrapure water (Direct-Q^®, Merck Millipore, Darmstadt, Germany) using a dialysis membrane (MWCO: 6000–8000 Da, Spectrum Lab, Rancho Dominguez, CA,USA) for 7 days and then lyophilized. Gelatin solution (5% w/v, in water) was added slowly in ADA solution (5% w/v, in phosphate buffered saline (PBS)) under continuous stirring to facilitate crosslinking between ADA and gelatin.
To prepare alginate solution, sodium alginate 2% (w/v) was dissolved in PBS and the prepared solution was sterilized by filtration using 0.45 μm filter.

Alginate dialdehyde-gelatin (ADA-GEL) beads were prepared at the Institute of Biomaterials, FAU Nuremberg-Erlangen as described elsewhere [12 (link)]. Briefly, sodium alginate derived from brown algae with a guluronic acid content of 65–70% (Sigma-Aldrich, Seelze, Germany) was oxidized using sodium metaperiodate (VWR International, Leuven, Belgium) in a 1:1 ethanol-water mixture. After 6 h, ethylene glycol (VWR International) was added to stop the reaction, and the solution was dialyzed against ultrapure water (Direct-Q^®, Merck Millipore, Darmstadt, Germany) using a dialysis membrane (MWCO: 6000–8000 Da, Spectrum Lab, Rancho Dominguez, CA, USA) for 7 days to remove unreacted periodate. After this, the solution was frozen and lyophilized. The lyophilized ADA was dissolved in PBS at a concentration of 5% (w/v). For the preparation of gelatin solution, gelatin powder from porcine skin (Bloom 300, Type A, Sigma-Aldrich, Seelze, Germany) was dissolved in ultrapure water at 37 °C at a concentration of 5% (w/v). ADA and gelatin solution were filtered through 0.45 μm and 0.22 μm filters (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) respectively, prior to mixing. Sterile ADA and gelatin were mixed in an equal volume ratio by slowly adding the gelatin solution into the ADA solution under continuous stirring to facilitate homogenous crosslinking.

LC–MS grade solvents were purchased from Merck Chemicals (Darmstadt, Germany) and high purity water was provided by a Millipore Direct-Q^® 3 UV purification system (MilliporeSigma, Burlington, MA, USA). Pierce™ LC–MS grade formic acid was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Gallic acid, Folin–Ciocalteu reagent and sodium carbonate were purchased from Sigma-Aldrich (St. Louis, MO, USA).