A colorimetric homogeneous immunoassay system for the C-reactive protein

Abstract

The C-reactive protein (CRP), which has a five repeat pentameric structure, is known to be a marker for acute inflammation and a potential risk predictor for cardiovascular disease. A simple and rapid homogeneous assay method for the detection of CRP, based on a gold nanoparticle (AuNP) aggregation induced colorimetric response, has been developed. In the technique, aggregation of CRP antibody-conjugated AuNPs is induced by addition of CRP as a consequence of its unique pentameric structure. CRP-promoted aggregation of the antibody-conjugated AuNPs results in a change of the wavelength maximum in the UV/Vis-spectrum. This homogeneous assay displays a typical hook effect, in which the signal level is directly proportional to CRP concentration until a critical concentration of CRP (the hook point) is reached. Above this concentration, the signal level decreases as the CRP concentration increases. The maximum shift in the absorption maximum was found to occur when the CRP antigen concentration is 100 ng mL⁻¹. In order to improve the linearity of the method, we employed a procedure, which takes advantage of a saturation phenomenon that leads to the hook effect, to increase the dynamic range of the CRP assay. Specifically, the use of CRP pre-spiked serum promotes maximum aggregation at the low CRP concentrations and, as a result, leads to an increase in the dynamic range for CRP detection. The applicability of the new homogenous assay system was demonstrated by its utilization for qualitative analysis of CRP in serum samples. The combined observations made in this effort show that the method using CRP antibody-conjugated AuNPs is both rapid and simple and, consequently, it can potentially be applied to onsite diagnosis.