Increased system sensitivity using fluorescent based immunoassays on the Gyrolab® platform

Abstract: Immunoassays have become an essential tool in several fields of bioanalytics with tremendous advances in sensitivity and formats seen through the last three decades. Many diseases are today diagnosed solely based on biomarker concentrations evaluated through immunoassays. More biomarkers will be unravelled for diseases that have low serum concentrations once highly sensitive analyzes can be performed routinely, highlighting the importance of improved sensitivity. The aim of this project was to increase the sensitivity of detection on the Gyrolab immunoassay platform. This was done by optimizing the conditions for fluorescence and by reduction of light scattering in the system. Four red-emitting fluorophores were investigated and assays were performed using additives with the purpose to reduce solvent-assisted quenching by water, and to avoid scattering of light in the system’s column. Deuterated solvents and encapsulation strategies were employed to reduce deactivation processes caused by water, and refractive index matching was used to limit the refraction of light. By using heavy water in assay sensitivity was increased by 17-25% for two biomarkers and with the use of different fluorophores showing consistency for the method. With the use of a refractive index matching liquid, it was possible to increase the depth at which the maximum fluorescence intensity occurred three-fold, using confocal microscopy. However, implementing found advantages in assay proved to be difficult due to mediums being hydrophobic and viscous, highlighting the complexity of the microfluidic system.