Antibody fluorescein-doped silica nanobioconjugates for the ultrasensitive detection of prostate-specific antigen†

Abstract

Herein, we report the bioconjugation of anti-prostate-specific antigen polyclonal antibodies (pAb) onto the fluorescein-doped silica nanoparticles to detect prostate-specific antigen (PSA). Fluorescein-isothiocyanate (FITC), a fluorescent dye, was reacted with (3-aminopropyl)triethoxysilane (APTES) to form the FITC-APTES organosilane precursor. FITC-APTES was mixed with tetraethoxysilane (TEOS) to form fluorescent silica nanoparticles (FITC@SiO₂NPs) containing 3% and 6% of dye loading. The silica nanoparticles prevented the dye from leaching and promoted the fluorescent signal amplification for the detection of PSA. Phenylboronic acid (PBA) was coated onto the fluorescent silica nanoparticles for the oriented antibody immobilization via the boronate ester to form FITC@SiO₂-PBA-pAb. The fluorescent silica nanobioconjugates exhibited an emission peak at 518 nm, which was stable over time. A fluorescence sandwich-type immunoassay was used for the detection of PSA using FITC@SiO₂-PBA-pAb. Alkaline hydrolysis of the sensing nanobioconjugates afforded enhanced sensitivity by releasing FITC molecules. In buffer samples, the fluorescent immunosensor exhibited a linear correlation range from 2.0 pg mL⁻¹ to 50 ng mL⁻¹. The linear range was from 2.0 pg mL⁻¹ to 100 ng mL⁻¹ in newborn calf serum (representing real samples). The limit of detection (LOD) was 8.25 fg mL⁻¹ with a limit of quantification (LOQ) of 27.2 fg mL⁻¹ in PBS (pH 7.4) after NaOH dissolution. A fluorescence immunosensor was used to detect PSA in spiked newborn calf serum with NaOH dissolution. It exhibited an LOD value of 33.0 fg mL⁻¹ and LOQ value of 0.109 pg mL⁻¹. The developed fluorescence immunosensor showed high selectivity and specificity for PSA. The detection of prostate-specific antigen in newborn calf serum samples exhibited no matrix interferences.