2.2 Sandwich immunoassay based on the silica- and magnetic-nanoparticles for detection of Brucella
Sandwich immunoassays using nanoparticle-based biosensors have recently received increasing attention as promising approaches for introducing selective and sensitive diagnostic tools (Sun, Zhao, & Dou, 2016). Silica- and magnetic-nanoparticles show unique features that can be related to their hydrophobic surfaces, ability to change their surfaces with chemical groups, and easy separation by the magnetic force (Taheri et al., 2020).
Magnetic nanoparticles (MNPs) have also been explored for the specific characterization of pathogenic bacteria, as they show remarkable enzyme activity with extremely high stability and they can be mass-produced at low cost. Pathogen-specific receptors like antibodies are therefore conjugated on the surface of the nanoparticles and are utilized to identify bacteria (Le, Tran, & Kim, 2020).
Shams et al. designed an immunosensor for characterization ofBrucella abortus based on the blue-silica nanoparticles (SiNPs) and paramagnetic nanoparticles (PMNPs). The synthesized immunosensor was conjugated with a polyclonal antibody against B. abortus and added to the bacterial suspension. Sandwich structure of PMNPs B. abortus -blue-SiNPs was then formed and separated by a magnet. Using a spectrophotometer, the absorbance of the blue color released from the silica structure was measured alongside the visible color shift to assess the presence of the bacterial cells in the samples (Shams, Rahimian Zarif, Salouti, Shapouri, & Mirzaii, 2019).
In another research, Taheri et al. designed an immunosensor, based on the magnetic- and silica-nanoparticles for detection of B. abortus . Consequently, IgG1 was conjugated on the surface of MNPs to form MNP−IgG1, and on the other hand, IgG2 and horseradish peroxidase enzyme (HRP) molecules were conjugated on the silica nanoparticles (SNPs) to form HRP−SNP−IgG2. The MNP−IgG1 and HRP−SNP−IgG2 were then added to a sample containing B. abortus to form HRP−SNP−IgG2−B. abortus −IgG1−MNPs complex followed by isolation of the complex using a magnet. Thereafter, tetramethylbenzidine (TMB) was added to the mixture to perform chromogenic reaction and the production of the blue color considered as the presence of B. abortus in the solution. In a positive sample, a blue color was seen due to the peroxidase activity of HRP on its substrate H2O2and subsequent oxidation of TMB chromogen. Whereas, the result was different in the negative sample since no HRP (HRP−SNP−IgG2) molecule was present in the solution after applying magnet in the previous step (Taheri et al., 2020). Schematic detection of B. abortus is given in figure 2.
2.3 Fluorescence assay using the immune magnetic beads and quantum dots for detection of Brucella
Quantum dots (QDs) are nanocrystals made by semiconductor materials that present attractive photophysical properties (Salouti & Ahangari, 2014). QDs are exceptionally bright, photostable and have high quantum yield. These properties make them suitable for sensing applications (Tallury, Malhotra, Byrne, & Santra, 2010). Li et al. designed an immunosensor by using the immune magnetic beads (IMB) probe and quantum dots (QDs) – staphylococcal protein A (SPA) probe for the diagnosis of brucellosis. The IMB probe and serum were first mixed to let the antibodies against Brucellainteract with a multi-epitope fusion protein of Brucella outer membrane protein (rOMP) coated on the surface of the IMB probe. Thereafter, the fluorescence intensity from QDs which was enhanced significantly and correlated with the number of anti-Brucella antibodies was measured to determine the result. The steps of the experiment are shown in figure 3 (L. Li et al., 2017).