In this paper, we describe the labelling of antibodies by gold nanoparticles (AuNPs) with diameters of 10 and 60 nm with detection by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Additionally, the AuNPs labelling strategy is compared with commercially available labelling reagents based on MeCAT (metal coded affinity tagging). Proof of principle experiments based on dot blot experiments were performed. The two labelling methods investigated were compared by sensitivity and limit of detection (LOD). The absolute LODs achieved were in the range of tens of picograms for AuNP labelling compared to a few hundred picograms by the MeCAT labelling.
For the first time, the combination of molecularly imprinted polymer (MIP) technology with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is presented with focus on an optimization of the LA-ICP-MS parameters such as laser beam diameter, laser beam fluence, and scan speed using CdS quantum dots (QDs) as a template and dopamine as a functional monomer. A non-covalent imprinting approach was employed in this study due to the simplicity of preparation. Simple oxidative polymerization of the dopamine that creates the self-assembly monolayer seems to be an ideal choice. The QDs prepared by UV light irradiation synthesis were stabilized by using mercaptosuccinic acid. Formation of a complex of QD-antibody and QD-antibody-antigen was verified by using capillary electrophoresis with laser-induced fluorescence detection. QDs and antibody were connected together
via
an affinity peptide linker. LA-ICP-MS was employed as a proof-of-concept for detection method of two types of immunoassay: 1) antigen extracted from the sample by MIP and subsequently overlaid/immunoreacted by QD-labelled antibodies, 2) complex of antigen, antibody, and QD formed in the sample and subsequently extracted by MIP. The first approach provided higher sensitivity (MIP/NIP), however, the second demonstrated higher selectivity. A mixture of proteins with size in range 10–250 kDa was used as a model sample to demonstrate the capability of both approaches for detection of IgG in a complex sample.
Immunochemical methods are used not only in clinical practice for the diagnosis of a wide range of diseases but also in basic and advanced research. Based on the unique reaction between the antibody and its respective antigens, it serves to specifically recognize target molecules in biological complex samples. Current methods of labelling antibodies with elemental labels followed by detection by inductively coupled plasma mass spectrometry (ICP-MS) allow detection of multiple antigens in parallel in a single analysis. Using the laser ablation (LA) modality (LA-ICP-MS), it is also possible to monitor the spatial distribution of biogenic elements. Moreover, the employment of metal nanoparticle-labeled antibodies expands the applicability also to molecular imaging by LA-ICP-MS. In this work, conjugates of model monoclonal antibody (DO-1, recognizing p53 protein) with various metal nanoparticles-based labels were created and utilized in dot-blot analysis in order to compare their benefits and disadvantages. Based on experiments with the p53 protein standard, commercial kits of gold nanoparticles proved to be the most suitable for the preparation of conjugates. The LA-ICP-MS demonstrated very good repeatability, wide linear dynamic range (0.1–14 ng), and limit of detection was calculated as a 1.3 pg of p53 protein.
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