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).