Culture-Free Quantification of Bacteria Using Digital Fluorescence Imaging in a Tunable Magnetic Capturing Cartridge for Onsite Food Testing

Abstract

Accurate, onsite detection of pathogenic bacteria from food matrices is required to rapidly respond to pathogen outbreaks. However, accurately detecting whole-cell bacteria in large sample volumes without an enrichment step remains a challenge. Therefore, bacterial samples must be concentrated, identified, and quantified. We developed a tunable magnetic capturing cartridge (TMCC) and combined it with a portable digital fluorescence reader for quick, onsite, quantitative detection of Staphylococcus aureus. The TMCC platform integrates an absorption pad impregnated with water-soluble polyvinyl alcohol (PVA) with an injection-molded polycarbonate (PC) plate that has a hard magnet on its back and an acrylonitrile-butadiene-styrene case. An S. aureus-specific antibody conjugated with magnetic nanoparticles was used to concentrate bacteria from a large-volume sample and capture bacteria within the TMCC. The retention time for capturing bacteria on the TMCC was adjusted by controlling the concentration and volume of the PVA solution. Concentrated bacterial samples bound to target-specific aptamer probes conjugated with quantum dots were loaded into the TMCC for a controlled time, followed by attachment of the bacteria to the PC plate and removal of unbound aptamer probes with wash buffer. The captured bacteria were quantified using a digital fluorescence reader equipped with an embedded program that automatically counts fluorescently tagged bacteria. The bacterial count made using the TMCC was comparable to a standard plate count (R 2 = 0.9898), with assay sensitivity and specificity of 94.3 and 100%, respectively.

Advancements in magnetic nanoparticle-based biosensors for point-of-care testing

“…MNPs are particularly effective in recognizing whole-cell bacteria in large samples without the need for enrichment processes, concentration, identification, or quantification of bacterial samples. For example, in the concentration of bacteria in large samples, Kaushal et al ( Kaushal et al, 2022 ) developed a tunable magnetic capture box (TMCC) that combines Staphylococcus aureus-specific antibodies with magnetic nanoparticles in controlled liquid samples for magnetophoretic concentration and capture ( Figure 1 ). The size of the MNPs and the strength of the magnetic field determine the duration required to capture the bacteria.…”

“…The quantification process is conducted using a portable fluorescence reader based on digital imaging. Reprinted with permission from Kausha et al (2019) .…”