The detection limit is one of the most important performance parameters for bioanalytical techniques. Here we present a generic method to estimate the detection limit of biomolecular assays based on a step-by-step analysis of the assay procedure. Enzyme-linked immunosorbent assay (ELISA) is used here as an example; however, much of the information presented in this article may be applied to other types of biomolecular assays and analytical techniques. A clear understanding of what affects the detection limit can help researchers to evaluate different bio-analytical techniques properly, and to design better strategies to optimize and achieve the best analytical performance.
In this study, we investigated the interaction between five biorelevant molecules and citrate-capped gold nanoparticles using dynamic light scattering, ζ-potential analysis, UV-vis absorption spectroscopy, and transmission electron microscopy. The five biomolecules are bovine serum albumin (BSA), two immunoglobulin G (IgG) proteins, immunoglobulin M (IgM), and a polysaccharide molecule, hyaluronan. BSA, IgG, and IgM are high abundance proteins in blood. Hyaluronan is a major component of the extracellular matrix. An abnormal level of hyaluronan in blood is associated with a number of medical conditions including rheumatoid arthritis and malignancy. Five different interaction modes were observed from these molecules. While BSA and IgM interact with the gold nanoparticles by forming electrostatic interactions with the citrate ligands, IgG and hyaluronan adsorb to the nanoparticle metal core by displacing the citrate ligands. BSA, rabbit IgG, and hyaluronan formed a stable monolayer on the nanoparticle surface. Human IgG and IgM caused nanoparticle cluster formation upon interacting with the gold nanoparticles. For the first time, we discovered that hyaluronan, a highly negatively charged polyglycosaminoglycan, exhibits an exceptionally strong affinity toward the citrate-gold nanoparticles. It can effectively compete with IgG to adsorb to the gold nanoparticles. This finding has exciting implications for future research: the molecular composition of a protein corona formed on a nanoparticle surface upon mixing the nanoparticle with blood or other biological fluids may vary according to the pathological conditions of individuals, and the analysis of these compositions could potentially lead to new biomarker discovery with diagnostic applications.
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