Paper-based analytical device for fast colorimetric detection of total hemoglobin and free hemoglobin from human blood sample

Total hemoglobin (THb) and free hemoglobin (FHb) are biomarkers of anemia and hemolysis, playing a critical role in human health status. This study reports a paper-based device by field amplified sample stacking (FASS) using colorimetric method to quantify both THb and FHb. The detection of THb was based on the color intensity of the Hb stacking band, a limit of detection (LOD) of 11 g∙L−1 and a linear working range of 16 ∼ 140 g∙L−1 were achieved. With selective chromatic reaction of the stacking band with o-tolidine, the sensitivity of Hb detection was further enhanced by 2000 times, a LOD of 25.8 mg∙L−1 and a linear range of 30 ∼ 1000 mg∙L−1 was obtained for FHb. This paper-based device was successfully used for detection of high concentration THb and low concentration FHb from the same blood sample, and both meeting the requirements of clinical diagnosis. In addition, the feasibility of on-site plasma preparation and online integration with the device was also preliminarily investigated with a blood separation membrane. This paper-based device could be used as a new platform for developing fast and low-cost Hb point-of-care testing devices.