A novel colorimetric sensing platform for the detection of S. aureus with high sensitivity and specificity

Abstract

In this study, a novel colorimetric sensing platform was developed for the detection of S. aureus using dog immunoglobulin G (IgG) as the capture antibody and chicken anti-protein A immunoglobulin Y labeled with horseradish peroxidase (HRP-IgY) as the detection antibody. Dog IgG labeled with magnetic beads was used to capture S. aureus through the interaction between the Fc region of dog IgG and Staphylococcal protein A (SPA). HRP-IgY was introduced to recognize the residual SPA on the surface of S. aureus and to create a sandwich format, after which a soluble 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added. A stop solution was utilized to cease the enzymatic chromogenic reaction, and then optical density was read at 450 nm. Under optimal conditions, the proposed method displayed a low detection limit of 1.0 × 10³ CFU mL^-1 and a wide linear range of 3.1 × 10³ to 2.0 × 10⁵ CFU mL^-1. This detection method exhibited high specificity against other foodborne bacteria. The recovery rates ranged from 95.2% to 129.2%. To our knowledge, this is the first report to employ dog IgG and chicken IgY as an antibody pair to detect S. aureus. This technique exhibits high application potential for S. aureus monitoring in various kinds of samples.

Fig. 1. Schematic illustration of the novel colorimetric sensing platform for the detection of S. aureus (not to scale).

Fig. 2. Effects of experimental components on A/A₀ of the system. (A) Effects of the amount of IMBs addition on A/A₀. (B) Effects of the concentration of HRP-IgY on A/A₀. Three independent measurements were taken from three individual preparations for each condition. Error bars indicated the standard deviations.

Fig. 3. Calibration curve of absorbance intensity (450 nm) along with S. aureus concentration under the optimal conditions in PBS. Three independent measurements were taken from three individual preparations for each condition. Error bars indicated the standard deviations.

Fig. 4. The specificity study for the proposed method for S. aureus detection. Red bars indicated absorbance intensity (450 nm) (from left to right) for 2.0 × 10⁶ CFU mL⁻¹E. coli O157:H7, Salmonella, Listeria monocytogenes and Streptococcus agalactiae; E. coli O157:H7, Salmonella, Listeria monocytogenes and Streptococcus agalactiae (2.0 × 10⁶ CFU mL⁻¹) interfered with 2.0 × 10⁵ CFU mL⁻¹S. aureus; S. aureus (2.0 × 10⁵ CFU mL⁻¹). Three independent tests were performed on three individual preparations for each condition. Error bars indicated the standard deviations.