posted on 2014-08-27, 00:00authored bySushma Kumari, Basab B. Dhar, Chakadola Panda, Abhishek Meena, Sayam Sen Gupta
Peroxidase, such as horseradish peroxidase
(HRP), conjugated to antibodies are routinely used for the detection
of proteins via an ELISA type assay in which a critical step is the
catalytic signal amplification by the enzyme to generate a detectable
signal. Synthesis of functional mimics of peroxidase enzyme that display
catalytic activity which far exceeds the native enzyme is extremely
important for the precise and accurate determination of very low quantities
of proteins (fM and lower) that is necessary for early clinical diagnosis.
Despite great advancements, analyzing proteins of very low abundance
colorimetrically, a method that is most sought after since it requires
no equipment for the analysis, still faces great challenges. Most
reported HRP mimics that show catalytic activity greater than native
enzyme (∼10-fold) are based on metal/metal-oxide nanoparticles
such as Fe3O4. In this paper, we describe a
second generation hybrid material developed by us in which approximately
25 000 alkyne tagged biuret modified Fe-tetraamido macrocyclic
ligand (Fe-TAML), a very powerful small molecule synthetic HRP mimic,
was covalently attached inside a 40 nm mesoporous silica nanoparticle
(MSN). Biuret-modified Fe-TAMLs represent one of the best small molecule
functional mimics of the enzyme HRP with reaction rates in water close
to the native enzyme and operational stability (pH, ionic strength)
far exceeding the natural enzyme. The catalytic activity of this hybrid
material is around 1000-fold higher than that of natural HRP and 100-fold
higher than that of most metal/metal oxide nanoparticle based HRP
mimics reported to date. We also show that using antibody conjugates
of this hybrid material it is possible to detect and, most importantly,
quantify femtomolar quantities of proteins colorimetrically in an
ELISA type assay. This represents at least 10-fold higher sensitivity
than other colorimetric protein assays that have been reported using
metal/metal oxide nanoparticles as HRP mimic. Using a human IgG expressing
cell line, we were able to demonstrate that the protein of interest
human IgG could be detected from a mixture of interfering proteins
in our assay.