In Situ Pinpoint Photopolymerization of Phos-Tag Polyacrylamide Gel in Poly(dimethylsiloxane)/Glass Microchip for Specific Entrapment, Derivatization, and Separation of Phosphorylated Compounds

An improved method for the online preconcentration, derivatization, and separation of phosphorylated compounds was developed based on the affinity of a Phos-tag acrylamide gel formed at the intersection of a polydimethylsiloxane/glass multichannel microfluidic chip toward these compounds. The acrylamide solution comprised Phos-tag acrylamide, acrylamide, and N,N-methylene-bis-acrylamide, while 2,2′-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] was used as a photocatalytic initiator. The Phos-tag acrylamide gel was formed around the channel crossing point via irradiation with a 365 nm LED laser. The phosphorylated peptides were specifically concentrated in the Phos-tag acrylamide gel by applying a voltage across the gel plug. After entrapment of the phosphorylated compounds in the Phos-tag acrylamide gel, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was introduced to the gel for online derivatization of the concentrated phosphorylated compounds. The online derivatized DTAF-labeled phosphorylated compounds were eluted by delivering a complex of phosphate ions and ethylenediamine tetraacetic acid as the separation buffer. This method enabled sensitive analysis of the phosphorylated peptides.

Carbon Dot-Mediated Capillary Electrophoresis Separations of Metallated and Demetallated Forms of Transferrin Protein

Carbon dots (CDs) are fluorescent nanomaterials used extensively in bioimaging, biosensing and biomedicine. This is due in large part to their biocompatibility, photostability, lower toxicity, and lower cost, compared to inorganic quantum dots or organic dyes. However, little is known about the utility of CDs as separation adjuvants in capillary electrophoresis (CE) separations. CDs were synthesized in-house according to a ‘bottom-up’ method from citric acid or other simple carbon precursors. To demonstrate the applicability of CDs as separation adjuvants, mixtures of holo- (metallated) and apo- (demetallated) forms of transferrin (Tf, an iron transport protein) were analyzed. In the absence of CDs, the proteins were not resolved by a simple CE method; however, upon addition of CDs to the separation buffer, multiple forms of Tf were resolved indicating that CDs are valuable tools to facilitate the separation of analytes by CE. CE parameters including sample preparation, buffer identity, ionic strength, pH, capillary inside diameter, and temperature were optimized. The results suggest that dots synthesized from citric acid provide the best resolution of various different forms of Tf and that CDs are versatile and promising tools to improve current electrophoretic separation methods, especially for metalloprotein analysis.

Determination of Erythromycin and Haloperidol in Human Urine by Capillary Electrophoresis Online Coupled with Electrochemiluminescence Detection

This work developed a simple and sensitive method for the simultaneous determination of erythromycin (Ery) and haloperidol (Hal) in human urine by capillary electrophoresis with eletrochemiluminescence detection. Under optimized conditions, such as detection potential at 1.25 V, electrokinetic injection at 10 kV for 6 s, separation voltage at 10 kV, 15 mmol/L separation buffer with pH 6.5, 5 mmol/L Ru(bpy)32+ and 50 mmol/L phosphate buffer with pH 8.0 in the ECL cell, the linear concentration ranges for Ery and Hal were from 0.005 to 0.2 μg/mL and from 0.15 to 6.0 μg/mL, respectively. The detection limits (3σ) for Ery and Hal were 0.002 and 0.06 μg/mL, respectively. When the method was applied to determine Ery and Hal in human urine, the recoveries were 96.5% and 95.1% on an average, respectively.