Size-exclusion chromatography for the characterization of urinary extracellular vesicles.

In recent years, extracellular vesicles (EVs) have gained attention for their potential as biomarkers for the early diagnosis and treatment of various diseases. Traditionally, EV isolation has relied exclusively on ultracentrifugation. However, alternative enrichment methods such as size-exclusion chromatography (SEC) and polyethylene glycol-based precipitation have been introduced. This study utilized SEC as a characterization tool to assess the efficiency of EV isolation. Urinary EVs isolated from human urine using centrifugation (40,000 × g) were analyzed using an SEC column with a pore size of 1000 Å, an inner diameter of 7.8 mm, and a length of 300 mm. The EVs were detected sequentially using UV (280 nm) and fluorescence (λex/em = 550 nm/565 nm); the EVs were observed at approximately 6 min, while the proteins were observed at approximately 12 min. The repeated centrifugation enrichment steps resulted in an increase in EV peaks and a decrease in protein peaks. SEC analysis of the enriched EV samples confirmed that a four-cycle repetition of centrifugation is necessary for successful EV enrichment and removal of non-EV proteins from 40 mL of human urine.

Aptamer-Based Single-Step Assay by the Fluorescence Enhancement on Electroless Plated Nano Au Substrate.

A new single-step aptamer-based surface-enhanced fluorescent optical sensor is built, by combining an aptamer-target interaction for target recognition and a fluorophore interaction for signal enhancement. The developed aptasensor is simple, sensitive, specific and stable for the detection of thrombin. A new nanometallic Au structure in the range of 100 nm was constructed through effective electroless plating method on a Cu thin film. Cu⁺ ions act as sacrificial seeds for the reduction of Au2+/3+ ions to form Au nanolawns. In order to utilize the structure for a fluorescence-based sensor, aptamer conjugated with Cy3 was immobilized on the nanogold substrate through electrostatic attraction. The Au substrate was coated with chitosan (molecular weight 1000 Da). Thrombin binding aptamer (TBA) was applied as a model system demonstrating the aptamer-based fluorescence assay on nanogold substrates. Thrice-enhanced fluorescence emission was achieved with Cy3-conjugated TBA stably immobilized on the chitosan-coated Au substrate. The intensity change was proportional to the concentration of thrombin from 10 µM to 10 pM, whereas the intensity change was ignorable for other proteins such as human serum albumin (HSA). Aptamer-based assay benefited from simple immobilization of receptors and Au nanostructure contributed in building an effective surface enhancing/positively charged substrate was proved. Such an aptasensor holding high utilities for point-of-care devices by incorporating simplicity, sensitivity and selectivity in detection, low-cost for test, small sample volumes has been developed.