In situ amplified photothermal immunoassay for neuron-specific enolase with enhanced sensitivity using Prussian blue nanoparticle-loaded liposomes

Methods based on prussian blue nanoparticles (PBNPs) have been reported for photothermal immunoassays in analytical nanoscience fields but most suffer from low sensitivity and are not beneficial for routine use. Herein, we design an in situ amplified near-infrared (NIR) photothermal immunoassay for the quantitative screening of neuron-specific enolase (NSE) on a portable thermometer using PBNP-encapsulated nanoliposomes as photosensitive materials. Biotinylated liposomes loaded with numerous prussian blue nanoparticles were synthesized through a typical reverse-phase evaporation method. The photothermal immunoassay was carried out in an anti-NSE capture antibody-coated microplate using the biotinylated anti-NSE secondary antibody. With the sandwiched immunoreaction and the biotin-avidin linkage, the subsequent photothermal measurement of PBNPs released from the liposomes with buffered surfactant including Tween 20 was conducted on a digital thermometer under near-infrared 808 nm laser irradiation, accompanied by the convertion of NIR-light wavelength to heat. Under the optimum conditions, the photothermal immunoassay displayed a wide dynamic concentration range of 0.1-100 ng mL-1 with a low detection limit for NSE of 0.053 ng mL-1. Good reproducibility (RSD ≤ 2.78% for intra-assay; RSD ≤ 4.39% for inter-assay), high selectivity against other biomarkers, and a long-term stability (≥94.9% of the initial signal during six-month storage) were acquired in the photothermal immunoassay. Impressively, the analysis of 7 human serum specimens for target NES via the photothermal immunoassay also gave well-matched results with the referenced human NSE enzyme-linked immunosorbent assay.