Exploitation of Luminescent Lanthanide Nanoparticles for a Sensitivity-Enhanced ELISA Detection Method

Abstract

A new detection method based on the photoluminescence properties of dye-sensitized lanthanide nanoparticles (Ln NPs) was developed for enzyme-linked immunosorbent assays (ELISAs). In this method, the horseradish peroxidase (HRP) enzyme catalyzes the oxidation of phenol derivatives in the presence of hydrogen peroxide, providing dimers that are able to interact with the Ln NP surface and to efficiently photosensitize the Ln ions. Due to the very long emission lifetime of Ln, the timegated detection of Ln NP luminescence allows the elimination of background noise due to the biological environment. After a comparison of the enzyme-catalyzed oxidation of various phenol derivatives, methyl 4-hydroxyphenyl acetate (MHPA) was selected as the most promising substrate, as the highest Ln emission intensity was observed following its HRP-catalyzed oxidation. After a meticulous optimization of the conditions of both the enzymatic reaction and the Ln sensitization (buffer, pH, concentration of the reactants, NP type, etc.), this new detection method was successfully implemented in a commercial insulin ELISA kit as a proof-ofconcept, with an increased sensitivity compared to the commercial detection method.

Structure–Activity Optimization of Luminescent Tb-doped LaF3 Nanoparticles

“…From this series, the 40% Tb-doped LaF 3 NPs arise as the best probe candidate, being among the most colloidally stable, monodisperse rounded NPs and exhibiting an average luminescence lifetime of 1.8 ms and the highest emission through DPA photosensitization. With improved luminescence properties compared to the already studied 10% Tb-doped NPs, such a luminescent probe would be very appealing to push forward the development of (bio)­analytical assays that require a higher sensitivity as obtained thanks to the background free time-resolved detection mode, for ELISA type assays , or for the detection of specific analytes depending on their coordination ability toward Ln atoms …”

“…When capped by appropriate light-harvesting coordinating ligands, they are highly luminescent and exhibit exceptional brightness up to 2.2 × 10 6 mol L –1 ·cm –1 along with a very long excited state lifetime (3.76 ms). Such NPs have been shown to be efficient luminescent probes for Förster resonance energy transfer (FRET)-based (bio)­sensing, immunoassays, and in vivo imaging . Various antennas have been tested to improve the luminescence properties of the NPs; however, the Tb doping rate is also an important factor to be considered.…”

Highly Specific Aptamer–Antibody Birecognized Sandwich Module for Ultrasensitive Detection of a Low Molecular Weight Compound

“…If the second antibody is linked to an enzyme in the enzyme-linked immunosorbent assay (ELISA), the presence of the target can be readily detected within dozens of minutes. , Thus, ELISA as a standardized diagnostic method has been widely used in the past decade due to simple operation, short time, and low cost. Despite these attributes, the false negatives caused by low valence of the antibody and the false positives caused by the complex environment of the sample reduce the credibility of ELISA. , Meanwhile, the enzymatic coloration reaction in the ELISA determines its detection limit at the nanomolar level, rendering ELISA already unable to meet the current demand. , More importantly, the assay is more suitable for the detection of macromolecular weight targets (proteins) rather than low molecular weight compounds (mycotoxins), since two antibodies with large steric hindrance are difficult to simultaneously bind to a small molecule target. Thus, the false negatives are significantly aggravated once the micromolecule is set as the target in ELISA. , Meanwhile, a nucleic-acid-based aptamer, a specific sequence recognizing a target molecule, can directly bind low molecular compounds via the flexible structure .…”