Gold nanoparticles bifunctionalized with antibodies and peroxidase for the detection of bacterial cells

Abstract

The enzyme immunoassay (EIA) is one of the most sensitive methods for detection of molecules (antigens) based on the use of specific antibodies and linked enzymes. In our opinion, the use of gold nanoparticles (AuNP) functionalized simultaneously by two types of protein molecules (antibody and enzyme) is a very promising direction in the development of EIA. The preparation of such bifunctional particles is simpler than chemical conjugations of two types of proteins, and the use is more efficient in comparison with standard antibody–AuNP conjugates. The aim of this work was to obtain stable AuNPs functionalized peroxidase and antibodies against a bacterial O-antigen.
In this work, we used solutions of peroxidase (Mw = 40 000 Da, 180 unit/mg; Dia-M LLC, Russia), antibodies to the cells of the bacterial strain Ochrobactrum cytisi IPA7.2. and AuNPs’ solution (d = 15 nm; 1.61012 particles/ml), which were provided by the staff of the laboratory of nanobiotechnology IBPPM RAS (Saratov).
At the first stage, the “golden number” for AuNPs and peroxidase solutions was determined by the salt method. Graphically, according to the dependence of the optical density of the mixture solution at 620 nm on the protein concentration, it was calculated that the "golden number" is 31.3 μg/ml. This corresponded to 32.7 peroxidase molecules per one AuNP. Next, a mixture of nanoparticles with 10 μg/ml peroxidase (concentration below the "golden number") was prepared, to which an equal volume of antibody solution (50 μg/ml) was added. After incubation during 15 minutes, the AuNPs were precipitated by centrifugation and dissolved in water. The resulting solution of nanoparticles was stable to salt aggregation, interacted with a specific antigen, and possessed enzymatic activity. The use of this conjugate in the dot assay demonstrated an 8-fold increase in the antigen detection sensitivity with the addition of a substrate solution for peroxidase as compared to staining with only functionalized AuNPs without enzyme reaction.


File with abstract

Speaker

Alina Kerimova
Saratov State University
Russia

Discussion

Ask question