Highly Sensitive Digital and Analog Immunoassay Based on Submicron Magnetic Beads and Fluorescent Microspheres

The detection of disease-related protein biomarkers plays a crucial role in the early diagnosis, treatment, and monitoring of diseases. The concentrations of protein biomarkers can vary significantly in different diseases or stages of the same disease. However, most of the existing analytical methods cannot simultaneously meet the requirements of high sensitivity and a wide dynamic range. Herein, we developed a digital and analog immunoassay method based on submicron magnetic beads and fluorescent microspheres. The digital analysis achieves a limit of detection as low as 46 fg/mL (1.8 fM) for IL-6, and the analog analysis has a wide dynamic range spanning from 0.2 pg/mL to 10 ng/mL. Furthermore, the analog analysis can be used to quickly and roughly assess higher concentration proteins via visual detection, and the practical application potential of this method was verified by the detection of alpha-fetoprotein in serum samples from 12 healthy individuals and hepatocellular carcinoma patients. The established method does not involve the use of enzymes or costly instruments, which greatly simplifies the experimental steps, shortens the experimental time, and reduces the experimental cost. In view of those advantages, the proposed method has great application prospects in point-of-care applications.