A colorimetric paper-based immunosensor for high-sensitivity C-reactive protein (hs-CRP) detection

Abstract

A simple colorimetric immunosensor using a paper-based analytical device (PAD) and a sandwich immunoassay was developed for the detection of high-sensitivity C-reactive protein (hs-CRP), a crucial risk predictor for cardiovascular diseases (CVDs). The capture antibody was immobilized onto magnetic beads to facilitate immunomagnetic separation (IMS) of CRP from the sample matrix, while the detection antibody was labelled with biotin. This biotinylated antibody subsequently linked to streptavidin conjugated with β-galactosidase (βgal), allowing a colorimetric assay due to the enzymatic reaction between βgal and chorophenol red-β-D-galactopyranoside (CRPG). The use of the highly sensitive CPRG substrate significantly enhanced the performance of the colorimetric hs-CRP detection with an LOD of 0.10 mg L⁻¹ and a detection range of 0.30–3.0 mg L⁻¹, which is clinically sufficient for the assessment of CVD risk. Furthermore, both intraday and interday measurements confirmed satisfied precisions with % RSD of less than 5%. Finally, the developed method was applied to determine CRP in a certified human serum reference material. The obtained result was found to be insignificantly different from the true value, confirming the reliability of the sensor. This simple, portable and instrument-free method, therefore, holds great promise for development as a screening test kit for CVD risk assessment in resource-limited settings.