Enzymatic Electrochemical Immunoassay Utilizing Magnetic Beads for Detection of Preterm Birth Biomarkers

Abstract

The mechanisms that preclude preterm birth are largely unknown. Studying the concentration changes of known preterm birth protein biomarkers in response to infections can help elucidate these mechanisms. The development of sensors for two potential biomarkers, matrix metalloproteinase 3 (MMP-3) and interleukin-1beta (IL-1β), will aid in such studies. IL-1β is an inflammatory cytokine that assists in initiating the inflammatory response associated with birth while MMP-3 plays an essential role in degrading the fetal membrane, inducing labor. Both IL-1β and MMP-3 are known to be present during a healthy birth, but when prematurely induced, can result in preterm birth.Magnetic bead-based competitive and sandwich assays are being developed to detect concentrations of IL-1β and MMP-3. These sensor designs utilize Horseradish peroxidase (HRP)/3,3′,5,5′-Tetramethylbenzidine (TMB) as the enzyme/substrate pair. Magnetic beads are modified with the antibody of interest and subsequently reacted with the target protein. In the competitive assays, the target protein is tagged with HRP; in the sandwich assays, a detection antibody is reacted with the target protein and subsequently tagged with HRP. When TMB is reacted with the modified beads, the HRP enzyme oxidizes TMB to TMB diimine which is then electrochemically reduced. The current generated in this process can be used to create a calibration curve with known protein concentrations versus current output. Current efforts are focused on improving sensitivity and replicability of the sensor designs. Future directions include monitoring the concentration of MMP-3 and IL-1 β before and after infection of an instrumental fetal membrane on-a-chip.