Theoretical Framework for Absolute Quantification in Digital Immunoassays and Advancements in Microfluidic-Free Droplet-Based Digital Immunoassay Methodologies

Conventional single-molecule immunoassay models, which model on a single discrete Poisson distribution, are inadequate for achieving theoretical absolute quantification of target proteins. We present a new method that models the capture of target proteins by magnetic beads in digital immunoassays as a Poisson process. In this approach, we treat the beads as discrete units and utilize the magnetic beads themselves as distinct objects to calculate the microvolume (Vd). The subsequent secondary sampling process is modeled using hypergeometric distributions. By implementing a droplet-based absolute quantification method, we establish two technical approaches, the bead-counting method and the external calibration method, that eliminate the necessity for traditional calibration curves. Comparative analyses with existing techniques, such as Single Molecule Immunoassays and Electrochemiluminescence Immunoassays, demonstrate enhanced feasibility and wide digital linear range of our methods. Furthermore, we explore the feasibility of an internal calibration method. Our theoretical framework effectively addresses the critical challenge of absolute quantification in protein immunoassays. It provides essential support for the development of precise protein quantification techniques and extends the capabilities of digital immunoassays in biomedical research and clinical diagnostics.