Gold nanoparticle (GNP)-based aggregation assay is simple, fast, and employs a colorimetric detection method. Although previous studies have reported using GNP-based colorimetric assay to detect biological and chemical targets, a mechanistic and quantitative understanding of the assay and effects of GNP parameters on the assay performance is lacking. In this work, we investigated this important aspect of the GNP aggregation assay including effects of GNP concentration and size on the assay performance to detect malarial DNA. Our findings lead us to propose three major competing factors that determine the final assay performance including the nanoparticle aggregation rate, plasmonic coupling strength, and background signal. First, increasing nanoparticle size reduces the Brownian motion and thus aggregation rate, but significantly increases plasmonic coupling strength. We found that larger GNP leads to stronger signal and improved limit of detection (LOD), suggesting a dominating effect of plasmonic coupling strength. Second, higher nanoparticle concentration increases the probability of nanoparticle interactions and thus aggregation rate, but also increases the background extinction signal. We observed that higher GNP concentration leads to stronger signal at high target concentrations due to higher aggregation rate. However, the fact the optimal LOD was found at intermediate GNP concentrations suggests a balance of two competing mechanisms between aggregation rate and signal/background ratio. In summary, our work provides new guidelines to design GNP aggregation-based POC devices to meet the signal and sensitivity needs for infectious disease diagnosis and other applications.

Vesicular assembly of small plasmonic nanoparticles, or plasmonic vesicle, is a promising multifunctional theranostic platform for photothermal therapy, near infrared (NIR) light‐responsive drug release, and rapid clearance of small inorganic particles from the body. Wide ranges of optical properties are reported including characteristic absorption peak in the visible or NIR ranges, or broadband absorption. It is unclear how the complex interaction among a large number of small gold nanoparticles contribute to the collective optical property of a plasmonic vesicle. In this study, the collective optical properties of plasmonic vesicles are examined and four characteristic regimes, namely the isolated nanoparticle regime, Coulomb interaction regime, black gold regime, and nanoshell regime, are revealed. Small plasmonic nanoparticles need to be very close or weakly overlap to give a broadband absorption (i.e., black gold regime) or form a NIR plasmon peak. Furthermore, smaller gold nanoparticle or larger core size leads to higher NIR peak shift and photothermal conversion efficiency. It is anticipated that this study provides design guidelines and thus have a significant impact on further design and development of complex plasmonic nanostructures and vesicles for biomedical applications.