Aptamer–antibody on-chip sandwich immunoassay for detection of CRP in spiked serum

Abstract

This study describes a RNA aptamer-based biochip with high affinity and specificity for C-reactive protein (CRP). CRP, which exists in concentrations of 1–3 mg/l in the serum of healthy patients, has been identified as a reliable biomarker for inflammation and as a potential marker for sepsis and tissue necrosis. The CRP-specific aptamer was covalently immobilized with its 5′-end on ARChip Epoxy. The detection of bound CRP was carried out optically using labelled secondary antibody in a sandwich format. Assay conditions were optimized with respect to the CRP binding buffer (buffer system, pH and additives) and Ca 2+ concentration (10 mM). Moreover, two sandwich immunoassay formats were tested, the one using dye-labelled antibodies and the other with biotin-modified antibodies/Dy647-labelled streptavidin. In comparison with an antibody-based chip assay, the aptamer chip is superior in terms of CRP measuring range (10 μg/l to 100 mg/l) in human serum whereas antibody-based chips result in superior data reproducibility (CV of 8–15%). In contrast to antibody chips, aptamer microarrays provide the unique potential of detecting CRP in serum samples of low risk patients (1–3 mg/l) as well as high risk patients (>500 mg/l), furthermore elevated CRP levels (20–350 mg/l) with acceptable recovery (70–130%) by including only one serum sample dilution step (1:100) for the complete measuring range.