Abstract
The purpose of this investigation was to study the effects of gold (Au) and silver (Ag) nanoparticles on a commonly used enzymatic reaction between horseradish peroxidase (HRP) and the substrate, 3,3',5,5'-tetramethylbenzidine (TMB). Two different methodologies were used in this study. The first was the addition of small quantities of nanoparticles, directly onto a solution of streptavidin peroxidase (streptavidin bound to HRP), prior to its reaction with TMB. The second, was the addition of nanoparticles to immobilised streptavidin peroxidase, covalently bounded to wells of a microtitre plate. In both cases, reactions with TMB were measured by visible absorbance spectroscopy, using a microtitre platereader. The results indicate that both gold and silver nanoparticles have a significant affect; with gold suppressing the reaction and silver enhancing it.
Highlights
The reaction between the enzyme, horseradish peroxidase (HRP), and the substrate, 3,3',5,5'-tetramethylbenzidine (TMB), is well established and frequently used in Enzyme-Linked Immunosorbent Assays (ELISAs) for detecting and measuring analytes, such as proteins and antibodies
A 2 μl drop of streptavidin peroxidase:phosphate buffered saline (PBS) solution was placed in the centre of one of the wells, which was used as a positive control reference well, i.e. without the addition of any nanoparticles
A 5 μl drop of 20 nm gold nanoparticles was placed in the centre of the second well, which was used as a negative control reference well, i.e. without the addition of any streptavidin:PBS solution
Summary
The reaction between the enzyme, horseradish peroxidase (HRP), and the substrate, 3,3',5,5'-tetramethylbenzidine (TMB), is well established and frequently used in Enzyme-Linked Immunosorbent Assays (ELISAs) for detecting and measuring analytes, such as proteins and antibodies. In the peroxidase, is combined with TMB, it produces a one-electron oxidation product, which is a free radical cation that is responsible for the change in colour from an almost colourless solution to dark blue as explained [1]. The degree of this colour change is directly reflective of the amount and activity of the enzyme; its use in ELISAs as the signal generating stage.
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