Enrichment of prostate-specific antigen using magnetic-silica antibody nanobioconjugates and fluorescence detection

Herein, we report the development of an immunosensor for the detection of prostate-specific antigen (PSA). The immunosensor platform was based on the immunometric sandwich protocol, using magnetic-silica nanoparticles for capture and pre-concentration of PSA. The preparation and application of the magnetic-silica nanobioconjugates and the use of phenylboronic acid for the immobilization of the capture antibody are the innovative steps of this report. The fluorescent sensing nanobioprobes contained 6% fluorescein in the fluorescein-doped silica nanoparticles. Silica nanoparticles can easily undergo alkaline dissolution for an enhanced fluorescence signal and thus ultrasensitive detection of PSA. The specificity of the immunosensor was achieved by the use of the anti-PSA monoclonal capture antibody (Ab1) bioconjugated in an oriented manner onto phenylboronic acid functionalized magnetic-silica nanoparticles. Non-specific binding sites were blocked with glucose to yield Fe3O4@SiO2-PBA-Ab1/glucose. Ab1 capture magnetic nanoparticles allowed for ease of separation using a magnet. For sensing, the polyclonal anti-PSA antibody (Ab2) was bioconjugated onto fluorescein-doped silica nanoparticles to form FITC@SiO2-PBA-Ab2/glucose. PSA was selectively isolated, enriched and purified from the serum samples using a magnetic nanobioconjugate (Fe3O4@SiO2-PBA-Ab1/glucose). A sandwich immunoreaction was achieved with FITC@SiO2-PBA-Ab2/glucose binding to the captured PSA. The alkali hydrolysis resulted in the disintegration of the nanoparticle thus releasing FITC molecules for fluorescence detection. This resulted in signal amplification. The analytical performance of the proposed immunosensor showed an excellent linear relationship between the fluorescence signal intensity and the concentration of PSA ranging from 2.0 pg mL-1 to 100 ng mL-1. The very low limit of detection (LOD) was 0.81 pg mL-1 and the limit of quantification (LOQ) was 2.46 pg mL-1. The immunosensor also exhibited good specificity and selectivity to PSA with 98.0-102.7% recovery rates. The detection was accomplished in newborn calf serum samples representing real samples.