Recent technological advancements in wearable sensors have made it easier to detect sweat components, but our limited understanding of sweat restricts its application. A critical bottleneck for temporal and regional sweat analysis is achieving uniform, high-throughput fabrication of sweat sensor components, including microfluidic chip and sensing electrodes. To overcome this challenge, we introduce microfluidic sensing patches mass fabricated via roll-to-roll (R2R) processes. The patch allows sweat capture within a spiral microfluidic for real-time measurement of sweat parameters including [Na+], [K+], [glucose], and sweat rate in exercise and chemically induced sweat. The patch is demonstrated for investigating regional sweat composition, predicting whole-body fluid/electrolyte loss during exercise, uncovering relationships between sweat metrics, and tracking glucose dynamics to explore sweat-to-blood correlations in healthy and diabetic individuals. By enabling a comprehensive sweat analysis, the presented device is a crucial tool for advancing sweat testing beyond the research stage for point-of-care medical and athletic applications.

Enzymatic conversion of polysaccharides into lower-molecular-weight, soluble oligosaccharides is dependent on the action of hydrolytic and oxidative enzymes. Polysaccharide monooxygenases (PMOs) use an oxidative mechanism to break the glycosidic bond of polymeric carbohydrates, thereby disrupting the crystalline packing and creating new chain ends for hydrolases to depolymerize and degrade recalcitrant polysaccharides. PMOs contain a mononuclear Cu(II) center that is directly involved in C-H bond hydroxylation. Molecular oxygen was the accepted cosubstrate utilized by this family of enzymes until a recent report indicated reactivity was dependent on HO Reported here is a detailed analysis of PMO reactivity with HO and O, in conjunction with high-resolution MS measurements. The cosubstrate utilized by the enzyme is dependent on the assay conditions. PMOs will directly reduce O in the coupled hydroxylation of substrate (monooxygenase activity) and will also utilize HO (peroxygenase activity) produced from the uncoupled reduction of O Both cosubstrates require Cu reduction to Cu(I), but the reaction with HO leads to nonspecific oxidation of the polysaccharide that is consistent with the generation of a hydroxyl radical-based mechanism in Fenton-like chemistry, while the O reaction leads to regioselective substrate oxidation using an enzyme-bound Cu/O reactive intermediate. Moreover, HO does not influence the ability of secretome from to degrade Avicel, providing evidence that molecular oxygen is a physiologically relevant cosubstrate for PMOs.

Factor Inhibiting Hypoxia–Inducible Factor (FIH) is an α-ketoglutarate (αKG) dependent enzyme which catalyzes hydroxylation of residue Asn803 in the C-terminal transactivation domain (CAD) of hypoxia–inducible factor 1α (HIF-1α) and plays an important role in cellular oxygen sensing and hypoxic response. Circular dichroism (CD), magnetic circular dichroism (MCD) and variable–temperature, variable–field (VTVH) MCD spectroscopies are used to determine the geometric and electronic structures of FIH in its (FeII), (FeII/αKG) and (FeII/αKG/CAD) forms. (FeII)FIH and (FeII/αKG)FIH are found to be six-coordinate (6C), whereas (FeII/αKG/CAD)FIH is found to be a 5C/6C mixture. Thus, FIH follows the general mechanistic strategy of nonheme FeII enzymes. Modeling shows that when Arg238 of FIH is removed the facial triad carboxylate binds to FeII in a bidentate mode with concomitant lengthening of the FeII–αKG-carbonyl bond, which would inhibit the O2 reaction. Correlations over α-keto acid-dependent enzymes and with the extradiol dioxygenases shows that members of these families (where both the electron source and O2 bind to FeII) have a second-sphere residue H-bonding to the terminal oxygen of the carboxylate, which stays monodentate. Alternatively, structures of the pterin-dependent and Rieske dioxygenases, which do not have substrate binding to FeII, lack H-bonds to the carboxylate, and thus allow its bidentate coordination which would direct O2 reactivity. Finally, Vis-UV MCD spectra show an unusually high-energy FeII→αKG π* metal-to-ligand charge transfer transition in (FeII/αKG)FIH which is red-shifted upon CAD binding. This red shift indicates formation of H bonds to the αKG that lower the energy of its carbonyl LUMO, activating it for nucleophilic attack by the Fe-O2 intermediate formed along the reaction coordinate.