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The low limit of detection (LLD) plays an important role in biomolecular assays, especially for early-stage assays. Biomolecular detections usually involve the use of two main elements: a reagent and an analyzer, which both greatly contribute to the LLD. In this work, the relationships among the LLD and reagent-related factors are investigated. The to-be-detected biomolecule is c-reactive protein (CRP) as an example. The assay method is immunomagnetic reduction (IMR). The components of reagent are Fe3O4 magnetic nanoparticles bio-functionalized with antibodies against CRP, dispersed in pH-7.4 phosphate buffered saline solution. Several key factors of the reagent, such as particle concentration, volume ratio of reagent to sample, and particle size, are manipulated to optimize the LLD of detecting CRP.