摘要： Improving the limit of detection by preconcentration and reducing the response time of optical biosensors are key requirements to enable their use in point of care (PoC) applications. To address these requirements, we have shown that integration of isoelectric focusing (IEF) at a pH step with a leaky waveguide (LW) sensor containing a non-speci?c a?nity ligand (reactive blue 4 dye (RB4)) can reduce the limit of detection of an exemplar protein (bovine serum albumin (BSA)) by a factor of 600–930 and reduce the response time to < 60 s. This is 6–9 times better preconcentration and up to 16 times faster response time than previous reports. IEF was performed with standard ampholytes and with simple acids and bases forming a pH step. Using ampholytes gave good preconcentration, but was much slower than using a pH step. The LW sensor used a thin agarose hydrogel layer into which RB4 was immobilized. The dye acted both as a non-speci?c a?nity ligand and to visualize the waveguide resonances. This allowed the refractive index of the waveguide to be monitored in real time at any point along the 10 mm separation channel length.