Illustration of a microtiter plate with different fluorescent solutions, which are used for the optical detection of inflammation markers.
Source: BAM
Immunoassays are used to detect molecules such as disease-relevant biomarkers or pollutants in the environment and in medical diagnostics. This bioanalytical test method is based on the specific binding between the molecule to be detected and antibodies, which have a high specificity for the target molecule. In most immunoassays, either the molecule or the antibody is labelled with a reporter in order to generate a measurable signal, which is then read out using optical methods, for example. The most common approach is the labelling with enzymes, which catalyse the conversion of an added substrate into an optically readable product. Direct labelling with fluorescent dyes is now playing an increasingly important role in simplifying such assays. The concentration of the target molecule in the sample is then determined via the measured signal intensity of the reporter. For the detection of very low concentrations, the signal intensity must be as high as possible for reliable measurements and quantifications.
Various signal amplification strategies for fluorescent reporters have been developed for this purpose. In our work, we have optimised and compared these strategies for the detection of the inflammatory marker C-reactive protein (CRP). A key to high signal strength is the labelling with a large number of reporter molecules contained in a nanoparticle bound to an antibody. However, the high loading density within the nanoparticle can lead to a quenching of the fluorescence intensity. Our new approach here is to release the reporter molecules after successful binding of the target molecule with the antibody by dissolving the nanoparticle. Parameters such as reporter concentration in the nanoparticle and the particle size are decisive for the highest possible signal amplification and very low detection limits.
This principle was investigated for the fluorescent reporter coumarin 153 and for hemin. Hemin catalyses a chemical reaction that leads to the fluorescence of the dye luminol. With the new signal amplification strategy, the latter approach enabled detection limits comparable to those of the enzyme-linked immunosorbent assay (ELISA) used in clinical studies. However, the approach we developed is faster and simpler and designed for reliable measurements outside of highly specialised laboratories.
Exploring Simple Particle-Based Signal Amplification Strategies in a Heterogeneous Sandwich Immunoassay with Optical Detection
Daniel Geißler, K. David Wegner, Christin Fischer, and Ute Resch-Genger
Analytical Chemistry 2024 96 (13), 5078-5085