Near-Infrared Fluorescence Immunoassay Using Streptavidin-Conjugated Oxygen-Doped Carbon Nanotubes

Abstract

Single-walled carbon nanotubes (CNTs) have been introduced to near-infrared (NIR) immunoassay systems for recent years. NIR lights have low absorption and scattering of biological matter and avoid autofluorescence from blood. Compared with other NIR molecules and materials, CNTs have several advantages such as strong emissions, large Stokes-shifts and high photo-stabilities as NIR labels. Previously, our research group reported that immunoprecipitation (IP) was successfully conducted between IgG-conjugated CNTs and protein G magnetic beads. Although the system can ideally measure target analytes at concentrations as low as 600 pmol/L, the practical reaction efficiency was insufficient, around ~18% [1] .To improve the IP reaction yield, we here made the streptavidin-conjugated oxygen-doped CNTs (o-CNT-SA) that can selectively bind with biotin molecules. The o-CNTs [3] were coated by streptavidin which combined with phospholipid polyethylene glycol (SA-PEG). While IgG forms a 1:1 complex with an antigen, SA can bind biotin molecules at four sites. The strength of streptavidin-biotin interaction is larger than antigen-antibody interaction, therefore it expects that the reaction efficiency become larger.Some amounts of phospholipid polyethylene glycol (PL-PEG) were added to stabilize the dispersion state of o-CNT-SA. For assessing the IP reactivity, the fluorescent intensity ratio between the eluted solution and the original o-CNT-SA dispersion was calculated (Fig. 1(a)). Figure 1(b) shows the relationship of the reaction efficiency and SA-PEG/PL-PEG concentrations. The contour lines indicate the reaction efficiency. At around ~50 µg/mL of SA-PEG concentration, the reaction efficiency shows the maximum. On the other hand, the reaction efficiency is better when the PL-PEG concentration is low while o-CNTs aggregate occurs at the PL-PEG concentration of ~144 µg/mL. Figure 1(c) shows the NIR fluorescence spectra observed from the eluted and the original o-CNT-SA solutions. By adjusting the concentrations of o-CNTs, SA-PEG and PL-PEG, the IP reaction yield increased up to 79.3 %. In this condition, molecular number ratio of o-CNT and SA-PEGs was estimated to be 1 : 11 – 46. Comparing the length of o-CNT, 500 nm, with the diameter of streptavidin, 8.2 nm, it expects that SA-PEGs adsorb on almost all surface of o-CNT. In the poster, we will show the detailed experimental procedures and discuss the proper structure of the o-CNT-SA NIR label based on the obtained results.[1] Y. Iizumi et al., ACS Appl. Mater. Interfaces, 5, 7665 (2013).[2] Y. Iizumi et al., Sci. Rep., 8, 6272 (2018). Figure 1