Publication details
Electrophoretic Purification Of Photon-Upconversion Nanoparticle Bioconjugates For A Single Molecule Upconversion-Linked Immunosorbent Assay
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| Year of publication | 2017 |
| Type | Conference abstract |
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| Citation | |
| Description | Counting individual molecules in a sample allows for a sensitive and straightforward determination of analyte concentration. Single molecule immunoassays with enzymatic signal amplification have been already reported. Luminescent nanoparticles recently emerged as an alternative molecular reporter avoiding the need for enzymatic substrate conversion. Photonupconversion nanoparticles (UCNPs) as background-free luminescent labels are particularly suitable for this purpose. UCNPs are lanthanide-doped nanocrystals that can be excited by near-infrared light and emit light of shorter wavelengths (anti-Stokes emission), which strongly reduces autofluorescence and light scattering. Further advantages of UCNPs include a very high photostability, large anti-Stokes shifts allowing for an excellent separation of excitation and detection channels, and multiple and narrow emission bands. For single molecule applications, homogeneous UCNP conjugates are required. However, UCNP conjugates are prone to aggregation, crosslinking, and nonspecific interactions. Therefore, a better control of the conjugate structure and surface modification is needed. Previously, we reported gel electrophoresis as a method for the analysis of silica coated UCNPs aggregation and purification of monodisperse nanoparticles. Here, this approach is extended for the analysis and the preparative purification of monodisperse UCNP conjugates, which are also suitable for single molecule immunoassays. We have developed an optical approach for visualizing individual UCNPs by conventional epiluminescence microscopy and applied it for a single molecule upconversion-linked immunosorbent assay (ULISA). As a model system, we have established a competitive ULISA for the sensitive detection of pharmaceutical micropollutant diclofenac at the single molecule level. The performance and sensitivity of the single molecule ULISA was critically dependent on the surface architecture of the luminescent tracer consisting of a diclofenac-UCNP conjugate. The tracer consisted of bright UCNPs (NaYF4: 18% Yb3+, 2% Er3+, ~90nm in diameter) embedded in a carboxylated silica shell, which was coated with diclofenacmodified bovine-?-globulin. Preparative gel electrophoresis immediately followed by electroelution was utilized for the purification of the tracer. A monodisperse conjugate was collected and utilized for diclofenac single molecule immunoassay. The purified monodisperse tracer allowed for homogeneous imaging and counting of individual UCNPs as diffraction limited spots. The limit of detection (LOD) for diclofenac was 0.15 ng mL-1. In contrast, the polydispersity of non-purified diclofenac-UCNP conjugates resulted in inhomogeneous spots of UCNPs and a lower number of UCNPs attached to the microtiter plate, which was not suitable for analytical applications. |
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