Novel UV-activated biofunctionalization of up-converting nanocrystals for detection of proteins

Abstract

Lanthanide doped nanocrystals capable to emit higher energy photons under excitation with lower energy radiation are promising for a broad range of applications including biodetection, biosensing, and bioimaging. However, the adaptation of these nanoparticles to the biological environment that requires good water-solubility, stability and ease of further functionalization still remains a challenge. The application of nanoparticles for biodetection or in various assays encountered many difficulties arising mainly from the strong tendency of nanoparticles for aggregation or nonspecific binding. Here we present a new method to obtain soluble and stable in water-based buffers NaYF4:Yb3+Er3+ nanocrystals with modified surface ready for further conjugation with biomolecules. In the presented approach polyvinylpyrrolidone/vinyl alcohol copolymer (PVP/VA) with photo-activatable linker (N-5-Azido-2-nitrobenzoyl group—ANB-NOS) was used for initial coating due to its high non-covalent affinity to nanoparticles surface. Subsequent coating with aminated dextran by ultraviolet light activation of ANB-NOS was carried out. This step has a significant impact on nanocrystals stability in the physiological buffer as well as on protein conjugation. Conjugation of biomolecules was possible by employing another photo-linker—sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA). Bacterial Protein G has been selected to create a universal immune-imaging agent due to its ability to bind Fc fragment of most mammalian immunoglobulins. Moreover, the direct attachment of antibodies to nanoparticles was also examined. The activity of biofunctionalized nanocrystals was tested in immunoblot test, that confirmed preserved activity of attached molecules and lack of undesired nonspecific binding or precipitation on the assay membrane. Graphic abstract

Upconversion Nanoparticle-Based Dot-Blot Immunoassay for Quantitative Biomarker Detection

“…In addition, Misiak et al reported on the use of UCNP labels in dot-blot immunoassays, however, focusing on different analytes. In their work, UCNPs were successively coated with a polyvinylpyrrolidone/vinyl alcohol copolymer, aminated dextran, and protein G. The prepared labels were then employed in semiquantitative detection of various targets, in particular, lipopolysaccharide from Escherichia coli (LOD of 1.95 μg/mL), murine monoclonal antibodies (LOD of 0.19 μg/mL), and immunoglobulin from human serum (LOD of 0.49 μg/mL).…”

“… 36 However, only one study has used UCNPs as labels for the sensitive quantitation of analyte concentrations in dot-blot. Misiak et al 37 used dextran-coated UCNPs conjugated with protein G for the detection of various protein targets, such as murine monoclonal antibodies, obtaining the LOD of 0.19 μg/mL. However, this study was not able to achieve sub-ng/mL detection limits, which are necessary for the sensitive detection of many low-abundance clinical biomarkers (defined by Kim et al 38 as biomarkers with concentrations below 10 ng/mL).…”