Ultratrace detection of C-reactive protein by a piezoelectric immunosensor based on Fe3O4@SiO2 magnetic capture nanoprobes and HRP-antibody co-immobilized nano gold as signal tags

An ultra-sensitive multiplexed signal amplified piezoelectric immunosensor for the detection of C-reactive protein (CRP) in serum was developed. Firstly, a sandwich-type immunoreaction was made between the capture probe [silicon dioxide-coated magnetic Fe3O4 nanoparticles (MNPs) labeled with the primary CRP antibody (MNPs-CRP Ab1)], CRP (different concentrations), and signal tag [horseradish peroxidase (HRP) coupled with the HRP linked secondary CRP antibody co-immobilized on gold nanoparticles (AuNPs-HRP/HRP-CRP Ab2)]. The immunocomplex was subsequently exposed to 3-amino-9-ethylcarbazole (AEC) and hydrogen peroxide. MNPs-CRP Ab1 capture probe could not only improve the CRP enrichment to amplify the signal through magnetic separation, but also be readily immobilized on the electrode by an external magnet to prepare the sensor. The signal tag could immobilize vast HRP, which could catalyze and yield more AECʹs insoluble oxidation product on piezoelectric crystal surface, also resulting in the amplified change in frequency response (Δf = f − f0). Thus the multiplex signal amplification was achieved. The Δf was proportional to CRP concentration from 0.001 to 100 ng mL−1 with a low detection limit of 0.3 pg mL−1. The optimum conditions were studied in detail. The proposed method could provide a fast, simple, and sensitive way for the clinical analysis of ultratrace CRP in heart diseases.