Sensitive Detection of Staphylococcus aureus with Vancomycin-Conjugated Magnetic Beads as Enrichment Carriers Combined with Flow Cytometry

Abstract

A novel sandwich strategy was designed to detect Staphylococcus aureus. The strategy is based on an antibacterial agent that captures bacterial cells and a fluorescein-labeled antibody that acts as the signal-output probe. Vancomycin (Van), which exerts a strong antibacterial effect on Gram-positive bacteria, was utilized as a molecular recognition agent to detect pathogenic bacteria. To effectively concentrate S. aureus, we used bovine serum albumin (BSA) as the amplification carrier to modify magnetic beads (MBs), which were then functionalized with Van. To improve the specificity of the method for S. aureus detection, we adopted fluorescein isothiocyanate (FITC)-tagged pig immunoglobulin G (FITC-pig IgG) as the signal probe and the second recognition agent that bound between the Fc fragment of pig IgG and protein A in the surface of S. aureus. To quantify S. aureus, we measured the fluorescence signal by flow cytometry (FCM). The use of multivalent magnetic nanoprobes (Van-BSA-MBs) showed a high concentration efficiency (>98%) at bacterial concentrations of only 33 colony-forming units (CFU)/mL. Furthermore, the sandwich mode (FITC-pig IgG/SA/Van-BSA-MBs) also showed ideal specificity because Van and IgG bound with S. aureus at two distinct sites. The detection limit for S. aureus was 3.3 × 10 CFU/mL and the total detection process could be completed within 120 min. Other Gram-positive bacteria and Gram-negative bacteria, including Listeria monocytogenes, Bacillus cereus, Cronobacter sakazakii, Escherichia coli O157:H7, and Salmonella Enteritidis, negligibly interfered with S. aureus detection. The proposed detection strategy for S. aureus possesses attractive characteristics, such as high sensitivity, simple operation, short testing time, and low cost.

Metal Nanoparticles for Diagnosis and Therapy of Bacterial Infection

“…[69] Apart from fluorescence signals, SERS has also been introduced in combination with magnetic biosensors. [70] Zhang et al used a SERS-based magnetic method to detect target bacteria ( Figure 4B). [67] In this strategy, Au NPs that were modified with Raman probe molecules (4-mercaptobenzoic acid (4-MBA) and 5,5′-Dithio-bis (2-nitrobenzoic acid) (DTNB)) and aptamers acted as the SERS substrate.…”

Electrochemical immunosensor based on an antibody-hierarchical mesoporous SiO2 for the detection of Staphylococcus aureus

“…It can be proved that the antibody and the mesoporous material are covalently bonded through the amide bond, thereby improving the stability of the immobilization. 38,39…”

“…1 Therefore, a new method for detecting low concentrations of S. aureus is required. For many years, conventional detection methods have included plate counting, uorescence quantitative polymerase chain reactions, 2 enzyme-linked immunosorbent assays 3 and mediated isothermal amplication techniques. 4 These methods have been applied and approved; however, most methods are expensive and cannot meet the fast, accurate, and highsensitivity requirements owing to the time and labor requirements.…”

Protein Binding on the Surface of Magnetic Nanoparticles

“…On this basis, the protein–MNP interaction will influence its cellular behavior and play a crucial role in biological applications and biotoxicity. MNPs are also modified by chemical groups, such as amino, carboxyl, and avidin, for in vitro applications, such as immunomagnetic separation and bacterial capture . The protein–MNP interaction is a precondition for these in vitro applications.…”