We describe the positive effect of surface plasmon-coupled fluorescence emission (SPCE) on the detection of a signal from a surface immunoassay in highly absorbing or/and scattering samples. A model immunoassay using fluorescently labeled anti-rabbit antibodies that bind to rabbit immunoglobulin on a silver surface was performed, and the signal was detected in the presence of various highly absorbing and/or scattering solutions or suspensions, such as hemoglobin solution, plastic beads, and red blood cells. The results showed that a highly absorbing solution consisting of small molecules (dye, hemoglobin) attenuates the SPCE signal approximately 2–3-fold. In contrast, suspensions with the same absorption containing large particles (large beads, red blood cell suspension) attenuate the SPCE signal only slightly, approximately 5–10%. Also, a suspension of large undyed, highly scattering beads does not reduce the SPCE signal. The effects on the immunoassay signal of the sample background absorption and scattering, the size of the background particles, and the geometry of the experimental set-up are discussed. We believe that SPCE is a promising technique in the development of biosensors utilized for surface-based assays, as well as any assays performed directly in highly absorbing and/or scattering solutions without washing or separation procedures.
- fluorescence immunoassays
- surface plasmon-coupled emission
- background suppression
- red blood cells