Particle clustering and coating across scales: microfluidic and macrofluidic experiments

In this thesis, I study the self-assembly of monodisperse colloidal particles on liquid-liquid interfaces. Specifically, I examine the relevant parameters that govern the size of self-assembled clusters when they pass through a liquid-liquid interface. I first describe a millimeter length-scale self-assembly system, where I find that the number of particles within a sinking cluster is proportional to a power law of the dimensionless Bond number. I find that the sphere deposition geometry also plays an important role, where I observe distinctly different scaling for monolayer rafts in comparison to stacked sphere clusters. I then develop an analogous microfluidic self-assembly system, where I use a magnetic field gradient to self-assemble paramagnetic microparticles on an aqueous two-phase liquid-liquid interface. Here, I observe empirically that the number of particles within a microparticle cluster scales inversely with the magnetic Bond number.

Particle clustering and coating across scales: microfluidic and macrofluidic experiments

In this thesis, I study the self-assembly of monodisperse colloidal particles on liquid-liquid interfaces. Specifically, I examine the relevant parameters that govern the size of self-assembled clusters when they pass through a liquid-liquid interface. I first describe a millimeter length-scale self-assembly system, where I find that the number of particles within a sinking cluster is proportional to a power law of the dimensionless Bond number. I find that the sphere deposition geometry also plays an important role, where I observe distinctly different scaling for monolayer rafts in comparison to stacked sphere clusters. I then develop an analogous microfluidic self-assembly system, where I use a magnetic field gradient to self-assemble paramagnetic microparticles on an aqueous two-phase liquid-liquid interface. Here, I observe empirically that the number of particles within a microparticle cluster scales inversely with the magnetic Bond number.

645. Singulex Clarity Norovirus Assay (In Development) Provides Ultrasensitive Detection of Norovirus Genogroups I and II

Background Commercially available enzyme immunoassays (EIAs) for detection of norovirus antigen have poor sensitivity and are limited to use in investigations of a gastroenteritis outbreak. Hence, there remains a need for a standalone high-sensitivity assay that enables rapid and accurate detection of norovirus antigen. Methods The Singulex Clarity norovirus assay is currently in development for use on the Singulex Clarity® system (Singulex Inc., Alameda, CA, USA), a fully-automated platform powered by Single Molecule Counting technology (registered with the FDA and CE marked). The assay uses paramagnetic microparticles bound to capture antibody and a fluorescently labeled reporter antibody to detect virion capsid protein of norovirus genogroups I (GI) and II (GII) in the stool. For the development of Clarity Norovirus assay, diagnostic performance of 4 antibody pairs (as Capture and Detection reagent) were evaluated by testing 137 stool samples from patients with suspected norovirus infection. Samples were sourced from three providers: (1) 90 genotyped samples of which 75 were positive (19 different genotypes) and 15 were negative by the CDC assay, (2) 3 samples positive and 5 samples negative by the BioFire® FilmArray® Gastrointestinal Panel, and (3) 39 samples negative by a lab-developed test using Cepheid reagents (SmartCycler®). Results From all the antibody pairs tested, one of the pairs had best performance with the area under the receiver operating characteristic (AuROC) curve demonstrating a C-Statistic of 0.959 (95% CI 0.921–0.997), compared with AuROC C-statistic of 0.943 (95% CI 0.896–0.990), 0.871 (95% CI 0.807–0.936), and 0.914 (95% CI 0.863–0.964) for the three other pairs. The Clarity assay detected all 19 different genotypes tested (figures). Conclusion The ultrasensitive and rapid Clarity norovirus assay (in development) for detection of GI and GII demonstrated excellent performance with one of the antibody pairs tested and detected all 19 tested genotypes. The Clarity assay may offer a standalone solution for norovirus diagnostics. Disclosures All authors: No reported disclosures.

Negative interference by rheumatoid factor in alpha-fetoprotein chemiluminescent microparticle immunoassay

Background Rheumatoid factor causes positive interference in multiple immunoassays. Recently, negative interference has also been found in immunoassays in the presence of rheumatoid factor. The chemiluminescent microparticle immunoassay is widely used to determine serum alpha-fetoprotein. However, it is not clear whether the presence of rheumatoid factor in the serum causes interference in the chemiluminescent microparticle immunoassay of alpha-fetoprotein. Methods Serum alpha-fetoprotein was determined using the ARCHITECT alpha-fetoprotein assay. The estimation of alpha-fetoprotein recovery was carried out in samples prepared by diluting high-concentration alpha-fetoprotein serum with rheumatoid factor-positive or rheumatoid factor-negative serum. Paramagnetic microparticles coated with hepatitis B surface antigen–anti-HBs complexes were used to remove rheumatoid factor from the serum. Results The average recovery of alpha-fetoprotein was 88.4% and 93.8% in the rheumatoid factor-positive and rheumatoid factor-negative serum samples, respectively. The recovery of alpha-fetoprotein was significantly lower in the rheumatoid factor-positive serum samples than in the rheumatoid factor-negative serum samples. In two of five rheumatoid factor-positive samples, a large difference was found (9.8%) between the average alpha-fetoprotein recoveries in the serially diluted and initial recoveries. Fourteen rheumatoid factor-positive serum samples were pretreated with hepatitis B surface antigen-anti-HBs complex-coated paramagnetic microparticles. The alpha-fetoprotein concentrations measured in the pretreated samples increased significantly. Conclusions It was concluded that the alpha-fetoprotein chemiluminescent microparticle immunoassay is susceptible to interference by rheumatoid factor, leading to significantly lower results. Eliminating the incidence of negative interference from rheumatoid factor should be an important goal for immunoassay providers. In the meantime, laboratorians must remain alert to the negative interference by rheumatoid factor, and in some cases, pretreat rheumatoid factor-positive samples with blocking or absorbing reagents.