A Low-Cost Micro-Volume Nephelometric System for Quantitative Immunoagglutination Assays.

Immunoassays have been widely used in scientific research and clinical diagnosis due to their versatile detection capability and high specificity. Immunoagglutination assays are kinds of immunoassay, which can simply and rapidly measure the concentration of analytes. In this work, we developed a low-cost micro-volume nephelometric system for quantitative immunoagglutination assays. We used off-the-shelf components to build the system, and the total cost of key components is only about 20 US dollars. The total detection volume in our system was as low as 3 µL, which could significantly reduce the reagent cost and required sample volume. We further evaluated the system performance via the immunoagglutination assay to measure the concentration of C-reactive protein, a plasma protein with levels rising in response to inflammation. The results demonstrated that our system could measure the concentration of analytes with relatively high sensitivity and precision within four minutes, and has high potential to be applied for clinical diagnostic tests.

Pyrroloquinoline quinone-doped polymeric nanospheres as sensitive tracer for binding assays.

The preparation and analytical characteristics of novel prosthetic group loaded polymeric nanospheres for use in high-sensitivity bioaffinity assays is reported. Pyrroloquinoline quinone (PQQ), a prosthetic group for apoglucose dehydrogenase (apo-GDH), is loaded into poly(methyl methacrylate) (PMMA) nanospheres in the presence of methanol. PQQ released from the nanospheres in the presence of 40% acetonitrile is capable of reconstituting apo-GDH and triggers the enzymatic reaction with excess glucose. The two electrons generated are transferred from a reduced PQQ to a redox dye reagent, e.g., 2,6-dichloroindolphenol (DCPIP). The decrease in absorbance of DCPIP is observed visually or spectrophotometrically to assess the number of particles present. As initial model systems, this concept is applied to develop a microtiter plate assay to detect biotin (as a model for low molecular weight species) and C-reactive protein (CRP). For the CRP assay, neutravidin-coated PQQ-doped PMMA nanospheres are used to bind with a biotinylated reporter antibody directed toward CRP. Detection limits to CRP at subnanogram per milliliter levels are demonstrated. The advantage of this type of assay is that excess apoenzyme can be added, with detection capability dependent on the number of encapsulated PQQ species that can be readily released from the surface-bound nanospheres (ca. 20,000 PQQ molecules/PMMA particle).