The use of bifunctional gold nanoparticles in enzyme-linked immunosorbent assay
Yulia A. Raspopova 1, Gennady L. Burygin 1,2; 1Saratov State University, Saratov, Russia; 2Institute of Biochemistry and Physiology of Plants and Microorganisms, Saratov Scientific Centre RAS, Saratov, Russia
Abstract
The enzyme-linked immunosorbent assay (ELISA) is one of the most widely used methods in laboratory diagnostics; however, conventional ELISA assays have limitations regarding sensitivity, assay duration, and reagent stability. A promising approach to improving the efficiency of ELISA is the use of gold nanoparticles (AuNPs) as carriers for antibodies (Ab) and enzyme labels.
In this study, spherical nanoparticles with a diameter of 15 nm were synthesized using the Frens method. To determine the “golden number” a series of two-fold dilutions of peroxidase (HRP) and Ab was prepared; these were added to AuNPs, then added NaCl measured the optical density at 690 nm. For both components, the “golden number” was 31.25 μg/mL.
The preservation of HRP enzymatic activity after immobilization was assessed separately for the pellet (conjugate) and the supernatant after centrifugation. A chromogenic substrate solution with o-phenylenediamine and H₂O₂ was added to the samples, and the optical density was measured at 492 nm. It was shown that immobilized HRP retains its catalytic activity, and that co-binding with Ab on the surface of AuNPs does not inhibit the enzyme. Bifunctional conjugates were obtained with four variants of the HRP:Ab mass ratio (ranging from 31.25 to 3.91 μg/mL HRP at a fixed Ab concentration). It was found that increasing the proportion of HRP enhances the analytical signal. Thus, the optimal ratio is determined by the specific application. The proposed approach allows for the design of conjugates with specified properties and can be adapted for a wide range of immunochemical assays.
Speaker
Yulia Raspopova
Saratov State University
Russia
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