Polymeric microsphere enhanced surface plasmon resonance imaging immunosensor for occult blood monitoring

Occult blood testing is a routine screening method for evaluating the function of the urinary system and digestive tract. The hemoglobin (Hb) content in human excreta is an important indicator of the presence of occult blood. In this study, a surface plasmon resonance imaging (SPRi)-based immunosensor with high sensitivity was developed for the detection of Hb. The immunosensor was constructed by physically immobilizing anti-Hb antibodies on the detection channels. Samples for evaluation were pre-incubated with the polymeric microsphere-antibody conjugate. The shift in resonance wavelength induced by the specific binding of microsphere-antibody-antigen and immobilized antibodies was monitored in real time. Owing to the amplification effect of microspheres, the detection limit of the immunosensor was reduced 42-fold to 4.8 ng/mL under the optimized concentration of immobilized antibody (10 μg/mL) with microsphere-antibody conjugate dilution ratio of 1:200 and incubation time of the microsphere-antibody conjugate and samples (10 min). The immunosensor displayed a good linear response to Hb ranging from 10 ng/mL to 500 ng/mL in both test solutions and in 1:30 urine sample, as well as a good specificity toward Hb and an acceptable recovery rate for spiked Hb solution in urine sample. The immunosensor was also exploited to detect the Hb concentration in 50 clinical urine samples. Therefore, the proposed SPRi immunosensor developed in this study provides a simple, rapid, high-throughput, and sensitive immunology strategy for the detection of occult blood.