Highly sensitive detection of C-reactive protein using a novel dissolution approach in a dye-doped silica nanoparticle-based fluorescence immunoassay

Abstract

A novel dissolution approach was used to achieve enhanced sensitivity in a nanoparticle (NP) immunoassay for the detection of a cardiovascular disease marker, C-reactive protein (CRP). In fluorescence-based assays using solid Cyanine5 doped silica nanoparticles, sensitivity enhancements of 32 times versus single dye labels were achieved. On release of the encapsulated dye from the particle at the assay end point by dissolution a further 128% increase in sensitivity was achieved as the previously partially self-quenched dye molecules were released. The limit-of-detection of the CRP assay using dissolved NPs was 20 times lower than for the single dye label due to a significant shift in the dynamic range. A CRP assay was also carried out using a biosensor device containing a CCD camera with laser diode excitation to demonstrate the feasibility of dissolution in a point-of-care application. In preliminary assays for the detection of human IgG significant improvements in assay precision and sensitivity versus a free dye label were also obtained. Prior to use, NP properties including size, dye loading and shell thickness were also optimised. The rate of change of fluorescence on dissolution was found to be zero-order with respect to the concentration of Cyanine5 inside the NP at any time. A simple model to explain zero-order kinetics was proposed based on changes in NP morphology on dissolution. These dissolvable NP labels have significant potential in medical diagnostics where cost is a limiting factor and clinical levels of sensitivity are not achievable using standard fluorophore labels. In addition, the zero-order kinetic behaviour has important implications for the potential use of NPs in drug delivery.