Development of Affinity Micro Free Flow Electrophoresis Immunoassay for Leptin

While existing immunoassays allow for measurements at discrete intervals, there are not yet refined affinity methods for continuous monitoring, leading to an incomplete understanding of biological processes. Micro free flow electrophoresis (µFFE) is a continuous analytical technique for separating, and quantifying analytes, which can help bridge this gap. Combining µFFE with affinity assays allows for the continuous monitoring and identification of antigens. With this technique, sample flows through a planar separation channel, and an electric field is applied orthogonally to the sample flow to separate analytes based on their electrophoretic mobility. Affinity µFFE allows for continuous detection and measurement of target analytes, eventually allowing for real-time detection of signaling peptides such as leptin to evaluate the effects of various stimuli.
Here, a COC µFFE device was fabricated via hot embossing and bonded using cyclohexane vapor exposure. A competitive µFFE immunoassay for leptin is being developed, in which fluorescein isothiocyanate (FITC) labeled leptin and unlabeled leptin compete for binding sites on anti-leptin antibody, followed by separation into two streams, one for leptin bound to antibody, and one for unbound leptin. Based on the fluorescent intensity of each stream the concentration of FITC-leptin that has bound to the anti-leptin antibody can be determined and subsequently the concentration of unlabeled leptin present can be determined. Injection of FITC-leptin demonstrates a low-nanomolar limit of detection, that the labeled antigen can be detected, and a linear calibration curve established. Preliminary offline experiments have been conducted using an anti-leptin antibody with FITC-leptin to verify binding, and a second peak can be detected. Further experiments are being conducted to establish a competitive immunoassay for leptin before applying the assay to adipose cells to develop the method into an online detection assay.