We demonstrate an all-electric sampling/derivatization/separation/recognition system for the quantitation of thiols

We demonstrate an all-electric sampling/derivatization/separation/recognition system for the quantitation of thiols in tissue cultures. cysteinefrom OHSCs within 25 s in a 23 mm separation channel with a confocal laser induced fluorescence (LIF) detector. Attention to the conductivities of the fluids being transported is required for successful flow-gated injections.When the sample conductivity is much higher than the run buffer conductivities the electroosmotic velocities are such that there is less fluid coming by electroosmosis into the cross from the sample/reagent channel than is leaving by electroosmosis into the separation and waste LIMK2 antibody channels. The resulting decrease in the internal fluid pressure in the injection cross pulls flow MRK 560 from the gated channel. This process may completely shut down the gated injection. MRK 560 Using a glycylglycine buffer with physiological osmolarity but only 62% of physiological conductivity and augmenting the conductivity of the run buffers solved this problem. Quantitation is by standard additions. Concentrations of cysteamine homocysteine and cysteine in the extracellular space of OHSCs are10.6±1.0 nM (n=70) 0.18 μM (n=53) and 11.1±1.2 μM (n=70) respectively. This is the first or in preparations such as acute or cultured brain slices. In this work we integrated online EO sampling and microfluidic analysis coupled to confocal LIF detection to evaluate the endogenous free cysteine homocysteine and cysteamineconcentrations in the extracellular space of OHSCs. Compared with publicationscited above the method described here is faster more sensitive; and to the best of our knowledge for the first time simultaneously evaluates the endogenous cysteine homocysteine and cysteaminein the extracellular space of any tissue. EXPERIMENTAL SECTION Chemicals and Reagents Tris (hydroxymethyl)aminomethane(Tris base) 1 3 propane) hydrochloric acid sodium hydroxide dimethyl sulfoxideanhydrous (DMSO) D-(+)-glucose glycylglycine L-cysteine DL-homocysteine cysteamine hydrochloride Gey’s balance salt solution (GBSS)and nitric acid (70%) MRK 560 were purchased from Sigma-Aldrich Chemical Co. (St. Louis MO).Hydrofluoric acid (48-50%) was fromFisher Scientific (Pittsburgh PA). Sodium chloride potassium chloride and MRK 560 methanolwere obtained from Avantor Performance Materials (Center Valley PA). Potassium phosphate monobasic and calcium chloride dihydrate werefrom EM Science (Gibbstown NJ). Opti-MEM horse serum propidium iodide (PI) and Hank’s balanced salt solution (HBSS) with phenol redwere from Life Technologies (Grand Island NY).ThioGlo-1 sodium bicarbonate and anhydrous magnesium sulfate were purchased from EMD Millipore (Billerica MA). Stock solutions ofcysteamine homocysteine and cysteinewere prepared no more than two days before experimentsusing degassed Milli-Q water (EMD Millipore) and stored at ?20 °C. The final concentrations of these solutions were 120.0 mM 69.1 mM and 49.7 mM individually. The stock solution of ThioGlo-1 was prepared using anhydrous DMSO with the final concentration of 2.5 mM. Stock solutions were diluted serially to the desired concentration during the experiments and kept on dry ice when not in use. The artificial cerebrospinal fluid (ACSF) was composed of 128 mM NaCl 3 mM KCl 2 mM CaCl2 1.2 mM MgSO4 0.4 mM KH2PO4 25 mM NaHCO3 and 10 mM D-glucose.42Glycylglycinemodified ACSF (ggACSF) was prepared by replacing 75 mM NaCl in ACSF with 125 mM glycylglycine and keeping all other components unchanged. Both solutions were adjusted to pH7.40 using NaOH. GBSS used during sampling experiments was MRK 560 fortified with 2.7 mM MgSO4 and 0.5% D-(+)-glucose.The term GBSS used below means “fortified GBSS” unless otherwise specified.PI solution was prepared using GBSS to a final concentration of 0.35 mM and was stored in the freezer. For microfluidic chip electrophoresis the run buffer was 40 mM Bis-Tris propane buffer with 15 mM NaCl (pH8.50) unless otherwise specified. Tris-HCl buffer (20 mM pH7.50)was used as the derivatizing buffer for the thiol analytes. All buffers were filtered through 0.1 μm polyethersulfone membrane (EMD Millipore) and degassed by ultrasonication prior to use. Design of a Microfluidic Chip Coupled withElectroosmotic Sampling The AZ 1500 photoresist coated borofloat glassplates (Telic.